Use of HSP70 as a regulator of enzymatic activity

ABSTRACT

The present invention concerns a method for modulating the enzymatic activity of an enzyme, wherein said enzyme interacts with BMP, said method comprising the step of administering or inducing Hsp70, or a functional fragment or variant thereof, in a form suitable for allowing interaction between BMP and Hsp70, or said functional fragment or variant thereof, and thereby modulating the enzymatic activity of an enzyme interacting with BMP.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/854,352, filed Dec. 26, 2017, now U.S. Pat. No. 10,543,204, which isa divisional of U.S. patent application Ser. No. 15/048,483, filed Feb.19, 2016, now U.S. Pat. No. 9,884,058, which is a divisional of U.S.patent application Ser. No. 13/969,944, filed Aug. 19, 2013, now U.S.Pat. No. 9,289,472, which is a continuation of U.S. patent applicationSer. No. 13/001,316, filed Dec. 23, 2010, now U.S. Pat. No. 8,540,985,which is a U.S. national phase of PCT/DK2009/050151, filed Jun. 26,2009, which is a non-provisional application of DK patent application PA2008 00885, filed Jun. 26, 2008. The entire content of each applicationis incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 19, 2019 isnamed “16ORPH-HO30708NA_ST25” and is 17.8 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of modulation of enzymeactivity by exploiting the interaction between the molecular chaperoneHsp70 and the lysosomal phospholipid Bis(monoacylglycero)phosphate (BMP,also known under the nome LBPA). The Hsp70-BMP interaction modulates theactivity of BMP-interacting enzymes of the lysosomal compartment, andthe present invention thus provides a means for reversing the pathologyof lysosomal storage diseases.

BACKGROUND OF THE INVENTION

The molecular chaperones are found in all compartments of a cell whereconformational rearrangements of proteins occur, and although proteinsynthesis is the major source of unfolded peptides in the cell, achallenge to the cell by high temperature or other stimuli that mightrender proteins structurally labile, and hence prone to unfolding andaggregation, is met with a specific cellular response involving theproduction of protective proteins. This response is a phenomenonobserved in every cell type ranging from prokaryotes to eukaryotes andis referred to as the heat-shock- or stress-response. The proteinsinduced by this response are known as the heat shock proteins (HSPS), ofwhich there exist several families.

A primary example of a family of chaperones is the Hsp70 proteins. Thisfamily has recently been implicated in other aspects of cellularhomeostasis besides serving as a chaperone—most markedly through itsanti-apoptotic features, its functions in immunity, and the apparentdependence of cancer cells on the upregulation of Hsp70. Furthermore,Hsp70 can serve a role in safeguarding lysosomal integrity. However, themolecular mechanism therefore has remained unclear.

The lysosomal storage diseases are a rare group of diseases,characterized by the accumulation of substances in the lysosomalcompartment and resulting destabilization hereof, with a resultingdevastating effect for affected individuals. Substances accumulate inthe lysosomal compartment due to deficiencies in the enzymes involved intheir catabolism.

To this date, no treatment is available for most lysosomal storagediseases. The underlying cause of this group of diseases is theinability of specific lysosomal enzymes to catabolize efficientlyspecific lysosomal substances such as lipids. Therefore the use ofenzyme replacement therapy (ERT), by providing to a patient therecombinant enzyme, has been employed for a subset of these diseases,including Gaucher and Fabry disease. However, ERT is a very expensiveform of therapy which may limit its use in some areas, and also iseffective only towards the specific type of disease to which therecombinant enzyme has been produced. The present invention is aimed atproviding new means for treating the lysosomal storage disorders.

SUMMARY OF THE INVENTION

In the present invention, the molecular basis for the contribution ofHsp70 to lysosomal membrane stability is disclosed by providing anunderstanding of the molecular basis for the association between Hsp70and cellular membranes—in particular plasma- and lysosomal membranes.

It is known from the literature that Hsp70 can serve a role insafeguarding lysosomal integrity. However, the molecular mechanism hasremained unclear. In addition, the question as to whether this attributeis specific for the major stress-inducible Hsp70 (HspA1A/A1B—named Hsp70throughout this study) or whether other Hsp70 family members could havethe same characteristic, had not been addressed either.

These unanswered questions prompted one of the major aims of thisinvention, which was to investigate the molecular basis for thelysosome-protective effect of Hsp70. To this end, a method for theproduction of recombinant Hsp70 and mutants hereof was set up, as was asubcellular fractionation protocol based on iodixanol gradientultracentrifugation. An assay for the direct assessment of lysososomalmembrane integrity was established based on photooxidation-inducedpermeabilization of lysosomes, which allowed a real-time microscopicapproach to evaluate the effect of Hsp70 and other components withregard to their ability to either sensitize or protect the lysosomalmembranes. The interaction of recombinant Hsp70 and mutants with variouslipids was investigated in different in vitro systems includingmeasurements of liposome 90° light scattering, tryptophan fluorescenceshifts and surface plasmon resonance (BIAcore). The creation of aconceptual model for the Hsp70-BMP interaction was aided by in silicoelectrostatic surface modeling of Hsp70. In order to verify the in vivorelevance of the lipid interaction witnessed in the in vitro systems,the BMP-Hsp70 interaction was targeted with regard to both components.To further show the feasibility of exploiting this mechanism, the modeof cell death induced by administration of cisplatin was characterized,and lysosomal Hsp70 was targeted in this cell death system both incancer as well as in non-transformed cell lines.

In order to address the molecular basis for Hsp70's contribution tolysosomal membrane stability, the inventors sought to establish a systemwhich would eliminate the influence of cytosolic Hsp70, i.e. targetingHsp70 directly to the lysosomes was needed. Electron microscopy picturesby Nylandsted et al. showed that Hsp70 could be present inside thelysosomes, and it was thus decided to establish a method for theproduction of recombinant human Hsp70 (rHsp70) and hopefully exploit theendocytic machinery as a delivery pathway of the rHsp70 directly to thelysosomes. The present inventors would hereby bypass the need for addinglysosomal sorting signals to Hsp70, potentially disrupting function andavoiding complications that might arise due to overexpression. Anendocytic approach would furthermore allow a titration of the amounts ofrHsp70 and in a longer perspective open possibilities for studying themechanism for uptake of extracellular Hsp70.

Having established the method for production of Hsp70, it was thentagged with the fluorophore Alexa Fluor 488 (Hsp70-AF488) in order tovalidate its endocytosis. Confocal imaging revealed that rHsp70 couldindeed be targeted to lysosomes in this way. In order to assess theimpact on lysosomal membrane stability, the inventors next set up amethod for quantifying lysosomal membrane permeabilization at the levelof single lysosomes and utilized this method to evaluate the effect ofendocytosed rHsp70. These methods formed the basis for Examples 1 and 2,in which the inventors show that Hsp70 enhances cell survival bystabilizing lysosomes through a pH-dependent high affinity binding tothe endo-lysosomal anionic phospholipid bis(monoacyl-glycero)phosphate(BMP). The positively charged ATPase domain of Hsp70 is responsible forthe binding but the substrate-binding domain is also required foreffective stabilization of lysosomes. Interestingly, this interaction,and the protection it offers, is dependent on tryptophan 90, which islocated in the positively charged wedge of the ATPase domain.Importantly, the cytoprotective effect could be obtained by endocyticdelivery of rHsp70 and specifically reverted by extracellularadministration of BMP antibodies or Hsp70 inhibitors.

In addition to this, the inventors also sought to couple the mechanismfor Hsp70's protection of lysosomal membranes to the events oftumorigenesis and programmed cell death. The inventors thuscharacterized the cell death program initiated by the administration ofa common chemotherapeutic agent, cisplatin and found it to beindependent of caspases, but characterized by lysosomal release ofproteases. Transgenic as well as endocytosed Hsp70 is capable ofenhancing cell survival in the face of cisplatin-challenge bystabilizing the lysosomal membranes. Interestingly, the inventors showthat either targeting lysosomal Hsp70 itself or its lysosomalinteraction partner bis(monoacyl-glycero)phosphate (BMP), sensitizetransformed, but not non-transformed, prostate cell lines to cisplatinwhich provides experimental evidence for exploiting the BMP-Hsp70interaction as a pharmacological target for cancer therapy.

Interestingly Hsp70-2, although sharing 86% amino acid homology withHsp70, was not capable of protecting the lysosomal membranes directly.However, the depletion of Hsp70-2 also results in lysosomal membranepermeabilization and ensuing programmed cell death. This effect does notdepend on a direct interaction between Hsp70-2 and the lysosomalcompartment, but is rather orchestrated via the down-regulation of LensEpithelium Derived Growth Factor (LEDGF), in response to Hsp70-2depletion.

The methods and results of this investigation are addressed in moredetail in the Examples section.

Having elucidated herein the molecular basis of the cytoprotectiveeffect of Hsp70 via an interaction with lysosomal BMP to promotelysosomal stabilization, these findings provide the basis for thetherapeutic targeting of lysosomal storage diseases.

It has now been demonstrated that surprisingly, providing recombinantHsp70 to cells efficiently reverts the pathology of lysosomal storagediseases, as shown herein for Niemann-Pick disease and Farber disease.Further, providing the Hsp70 inducer benzyl alcohol to cells efficientlyreverts the pathology of lysosomal storage diseases, as shown herein forNiemann-Pick disease.

The present invention thus provides a method for treating lysosomalstorage diseases by increasing directly or indirectly the intracellularconcentration and/or activity of Hsp70 in individuals in need thereof,by providing Hsp70, or a functional fragment or variant thereof, or byproviding a Hsp70 inducer or co-inducer.

The present invention relates in one aspect to a bioactive agent capableof increasing the intracellular concentration and/or activity of Hsp70for use as a medicament or for use in the treatment of a lysosomalstorage disorder.

In one embodiment, said bioactive agent is Hsp70, or a functionalfragment or variant thereof.

In another embodiment, said bioactive agent is an Hsp70 inducer orco-inducer.

It is also an aspect of the present invention to provide a method fortreatment of a lysosomal storage disease comprising administration ofthe bioactive agent according to the present invention to an individualin need thereof.

In one embodiment, said treatment is prophylactic, curative orameliorating.

In one embodiment, said lysosomal storage disease is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Sialidosis, Metachromaticleukodystrophy and saposin-deficiency.

In another embodiment, said lysosomal storage disease is characterisedas having residual enzymatic activity of the defective enzyme involvedin the disease pathology.

The present invention also relates to a method of treatment of alysosomal storage disease comprising administration of the bioactiveagent according to the present invention in combination with at leastone other treatment modality.

A further aspect of the present invention is to provide a method formodulating the enzymatic activity of an enzyme, wherein said enzymeinteracts with BMP (bis(monoacylglycero)phosphate), said methodcomprising the steps of

-   -   i) administering the bioactive agent according to the present        invention,    -   ii) allowing interaction between BMP and Hsp70, and    -   iii) modulating the enzymatic activity of an enzyme interacting        with BMP.

In another aspect, the present invention relates to Hsp70, or afunctional fragment or variant thereof, for use as a medicament.

In one aspect, the present invention concerns a method for modulatingthe enzymatic activity of an enzyme, wherein said enzyme interacts withBMP, said method comprising the step of administering Hsp70, or afunctional fragment or variant thereof, in a form suitable for allowinginteraction between BMP and Hsp70, or said functional fragment orvariant thereof, and thereby modulating the enzymatic activity of anenzyme interacting with BMP.

Preferably, Hsp70 or said functional fragment or variant thereof forms acovalent or non-covalent complex with BMP.

Preferably, BMP interacts with a saposin.

Preferably, said saposin is selected from the group consisting ofsaposin A, saposin B, saposin C, and saposin D.

Preferably, said enzyme is selected from the group consisting ofsphingomyelinase, acidic sphingomyelinase, sialidase,alpha-galactosidase, beta-galactosidase, beta-galactosylceremidase,glucosylceremidase, and acid ceremidase.

Preferably said modulation of the enzymatic activity is an up-regulationof the enzymatic activity of said enzyme.

In another aspect, the present invention concerns Hsp70, or a functionalfragment or variant thereof, for use as a medicament. Preferably, saidHsp70, or a functional fragment or variant thereof, may be used in thetreatment, alleviation, or prophylaxis of a lysosomal storage disorder,such as the disorders Niemann-Pick, Gaucher, Farber, Krabbe, Fabry, andSialidosis.

In another aspect, the invention concerns a method for increasing theuptake of a compound, said method comprising the step of administeringsaid compound together with Hsp70 or a functional fragment or variantthereof. In one embodiment, said Hsp70 or a functional fragment orvariant thereof is covalently bound to said compound. In anotherembodiment, said Hsp70 or a functional fragment or variant thereof isnon-covalently bound to said compound.

An embodiment of the invention concerns a method for up-regulation of anenzymatic activity of an enzyme associated with a lysosomal storagedisorder, such as Niemann-Pick, Gaucher, Farber, Krabbe, Fabry, andSialidosis. Preferably, said lysosomal storage disorder is Niemann-Pick.

Since the lysosomal storage disorders are caused by insufficientenzymatic activity, it is the aim of the invention to increase theenzymatic activity in order to alleviate or cure the disorder.

Hsp70 has been shown to interact with BMP. Since BMP acts as a co-factorfor various other proteins, the interaction between Hsp70 and BMP maymodulate the function of these various other proteins. For instance, BMPacts as a co-factor for aSMase. Thus, the interaction between Hsp70 andBMP may increase the activity of aSMase. Since Niemann-Pick disorder isassociated with a decreased aSMase activity, Hsp70 may alleviate or cureNiemann-Pick disorder by increasing the activity of aSMase. Similarly,BMP acts as a co-factor for the saposin A, saposin, B, saposin C, andsaposin D. These saposin proteins are implicated in other lysosomalstorage disorders, and therefore Hsp70 may alleviate or cure otherlysosomal storage disorders by increasing the activity of a saposin orof an enzyme associated with said saposin.

In an embodiment of the invention, Hsp70 is administered together withenzyme replacement therapy in the treatment of a lysosomal storagedisorder. In this manner, the amount of enzyme necessary may besignificantly reduced due to the enzyme-activating effect of Hsp70.

In another embodiment, Hsp70 is used to facilitate uptake of enzymes inenzyme replacement therapy, thereby increasing the amount of enzymehaving been taken up by the relevant cells.

Definitions and Abbreviations

-   aSMase/ASM Acidic sphingomyelinase-   ADD70: AIF-derived decoy for Hsp70-   AIF: Apoptosis inducing factor-   AO: Acridine Orange-   Apaf-1: Apoptotic protease activating factor-1-   Bag-1: Bcl-2 associated athanogene-1-   Bcl-2: B-cell lymphoma/leukaemia 2-   Bid: BH3 interacting domain death agonist-   BMP: Bis(monoacylglycero)phosphate-   CARD: Caspase recruitment domain-   Caspase: Cysteine aspartate-specific protease-   CHIP: Carboxy terminus of Hsp70-binding protein-   CytC: Cytochrome C-   DD: Death domain-   DED: Death effector domain-   dsRNA: double-stranded RNA-   eHsp70: extracellular Hsp70-   ER: Endoplasmic reticulum-   ERT Enzyme replacement therapy-   FADD: Fas-associated death-domain containing protein-   HIP: Hsp70 interacting protein-   HRP: Horse radish peroxidase-   HS: Heat shock/stress-   HSE: Heat shock element-   HSF: Heat shock factor-   Hsp: Heat shock protein-   HspBP1: Heat shock protein Binding Protein 1-   IAP: Inhibitor of apoptosis protein-   iMEF immortalized Murine Embryonic Fibroblasts-   JNK: c-jun NH2-terminal kinase-   LAMP-1/-2: Lysosome-associated membrane protein-1/-2-   LBPA: Lysobisphosphatidic acid-   LEDGF: Lens epithelium derived growth factor-   LMP: Lysosomal membrane permeabilization-   MIC-1: Macrophage inhibitory cytokine 1-   MOMP: Mitochondrial outer membrane permeabilization-   MPR Mannose 6-phosphate receptor-   MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium    bromide-   NPD Niemann-Pick disease-   NPDA Niemann-Pick disease, type A-   NPDB Niemann-Pick disease, type B-   NPDC Niemann-Pick disease, type C-   NPDD Niemann-Pick disease, type D-   PCD: Programmed cell death-   PKC: Protein kinase C-   POPC: Palmitoyl-oleoyl-phosphatidylcholine-   POPS: Palmitoyl-oleoyl-phosphatidylserine-   RNAi: RNA interference-   ROS: Reactive oxygen species-   SD: Standard deviation-   siRNA: Short interfering RNA-   Smac/Diablo: Second mitochondrial-derived activator of caspases-   tBid: Truncated Bid-   TNF: Tumour necrosis factor-   TNFR: TNF-receptor-   TRADD: TNFR associated death domain protein-   TRAF: TNFR associated factor

Lysosomal storage disorder (LSD): The terms “lysosomal storage disorder”and “lysosomal storage disease” are used as synonyms.

Functional fragment of Hsp70: The term “functional fragment of Hsp70” isto be construed as meaning any fragment of Hsp70 having the desiredfunction. In relation to modulation of enzymatic activity, a functionalfragment is a fragment capable of modulating the enzymatic activity. Inrelation to increasing the uptake of a substance, a functional fragmentof Hsp70 is a fragment capable of increasing the uptake of saidsubstance. It is appreciated that the exact quantitative effect of thefunctional fragment may be different from the effect of the full-lengthmolecule. In some instances, the functional fragment may indeed be moreeffective than the full-length molecule. Furthermore, the use offragments instead of full-length molecules may be advantageous in viewof the smaller size of the fragments.

Functional variant of Hsp70: The term “functional variant of Hsp70” isto be construed as meaning any variant of Hsp70 having the desiredfunction. In relation to modulation of enzymatic activity, a functionalvariant is a variant capable of modulating the enzymatic activity. Inrelation to increasing the uptake of a substance, a functional variantof Hsp70 is a fragment capable of increasing the uptake of saidsubstance. It is appreciated that the exact quantitative effect of thefunctional variant may be different from the effect of the full-lengthmolecule. In some instances, the functional variant may indeed be moreeffective than the full-length molecule.

A “Bioactive agent” (i.e., biologically active substance/agent) is anyagent, drug, compound, composition of matter or mixture which providessome pharmacologic, often beneficial, effect that can be demonstrated invivo or in vitro. As used herein, this term further includes anyphysiologically or pharmacologically active substance that produces alocalized or systemic effect in an individual. Further examples ofbioactive agents include, but are not limited to, agents comprising orconsisting of an oligosaccharide, agents comprising or consisting of apolysaccharide, agents comprising or consisting of an optionallyglycosylated peptide, agents comprising or consisting of an optionallyglycosylated polypeptide, agents comprising or consisting of a nucleicacid, agents comprising or consisting of an oligonucleotide, agentscomprising or consisting of a polynucleotide, agents comprising orconsisting of a lipid, agents comprising or consisting of a fatty acid,agents comprising or consisting of a fatty acid ester and agentscomprising or consisting of secondary metabolites. It may be used eitherprophylactically, therapeutically, in connection with treatment of anindividual, such as a human or any other animal. As used herein, abioactive agent is a substance capable of increasing the intracellularconcentration and/or activity of Hsp70.

The terms “drug” or “medicament” as used herein includes biologically,physiologically, or pharmacologically active substances that act locallyor systemically in the human or animal body.

The terms “treating”, “treatment” and “therapy” as used herein referequally to curative therapy, prophylactic or preventative therapy andameliorating or palliative therapy. The term includes an approach forobtaining beneficial or desired physiological results, which may beestablished clinically. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) condition, delay or slowing of progression or worsening ofcondition/symptoms, amelioration or palliation of the condition orsymptoms, and remission (whether partial or total), whether detectableor undetectable. The term “palliation”, and variations thereof, as usedherein, means that the extent and/or undesirable manifestations of aphysiological condition or symptom are lessened and/or time course ofthe progression is slowed or lengthened, as compared to notadministering compositions of the present invention.

A “treatment effect” or “therapeutic effect” is manifested if there is achange in the condition being treated, as measured by the criteriaconstituting the definition of the terms “treating” and “treatment.”There is a “change” in the condition being treated if there is at least5% improvement, preferably 10% improvement, more preferably at least25%, even more preferably at least 50%, such as at least 75%, and mostpreferably at least 100% improvement. The change can be based onimprovements in the severity of the treated condition in an individual,or on a difference in the frequency of improved conditions inpopulations of individuals with and without treatment with the bioactiveagent, or with the bioactive agent in combination with a pharmaceuticalcomposition of the present invention.

“Pharmacologically effective amount”, “pharmaceutically effectiveamount” or “physiologically effective amount of a “bioactive agent” isthe amount of an active agent present in a pharmaceutical composition asdescribed herein that is needed to provide a desired level of activeagent in the bloodstream or at the site of action in an individual (e.g.the lungs, the gastric system, the colorectal system, prostate, etc.) tobe treated to give an anticipated physiological response when suchcomposition is administered. The precise amount will depend uponnumerous factors, e.g., the active agent, the activity of thecomposition, the delivery device employed, the physical characteristicsof the composition, intended patient use (i.e. the number of dosesadministered per day), patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein. An “effective amount” of a bioactive agentcan be administered in one administration, or through multipleadministrations of an amount that total an effective amount, preferablywithin a 24-hour period. It can be determined using standard clinicalprocedures for determining appropriate amounts and timing ofadministration. It is understood that the “effective amount” can be theresult of empirical and/or individualized (case-by-case) determinationon the part of the treating health care professional and/or individual.

The terms “enhancing” and “improving” a beneficial effect, andvariations thereof, as used herein, refers to the therapeutic effect ofthe bioactive agent against placebo, or an increase in the therapeuticeffect of a state-of-the-art medical treatment above that normallyobtained when a pharmaceutical composition is administered without thebioactive agent of this invention. “An increase in the therapeuticeffects” is manifested when there is an acceleration and/or increase inintensity and/or extent of the therapeutic effects obtained as a resultof administering the bioactive agent(s). It also includes extension ofthe longevity of therapeutic benefits. It can also manifest where alower amount of the pharmaceutical composition is required to obtain thesame benefits and/or effects when it is co-administered with bioactiveagent(s) provided by the present invention as compared to theadministration in a higher amount of the pharmaceutical composition inthe absence of bioactive agent. The enhancing effect preferably, but notnecessarily, results in treatment of acute symptoms for which thepharmaceutical composition alone is not effective or is less effectivetherapeutically. Enhancement is achieved when there is at least a 5%increase in the therapeutic effects, such as at least 10% increase inthe therapeutic effects when a bioactive agent of the present inventionis co-administered with a pharmaceutical composition compared withadministration of the pharmaceutical composition alone. Preferably theincrease is at least 25%, more preferably at least 50%, even morepreferably at least 75%, most preferably at least 100%.

“Co-administering” or “co-administration” of bioactive agent(s), orbioactive agents and state-of-the-art medicaments, as used herein,refers to the administration of one or more bioactive agents of thepresent invention, or administration of one or more bioactive agents ofthe present invention and a state-of-the-art pharmaceutical compositionwithin a certain time period. The time period is preferably less than 72hours, such as 48 hours, for example less than 24 hours, such as lessthan 12 hours, for example less than 6 hours, such as less than 3 hours.However, these terms also mean that the bioactive agent and atherapeutic composition can be administered together.

The term “Individual” refers to vertebrates, in particular a member of amammalian species, preferably primates including humans. In a preferredembodiment, an individual as used herein is a human being, male orfemale, of any age.

An “individual in need thereof” refers to an individual who may benefitfrom the present invention. In one embodiment, said individual in needthereof is a diseased individual, wherein said disease is a lysosomalstorage disease.

The term “natural nucleotide” or “nucleotide” refers to any of the fourdeoxyribonucleotides, dA, dG, dT, and dC (constituents of DNA), and thefour ribonucleotides, A, G, U, and C (constituents of RNA), as found innature. Each natural nucleotide comprises or essentially consists of asugar moiety (ribose or deoxyribose), a phosphate moiety, and anatural/standard base moiety. Natural nucleotides bind to complementarynucleotides according to well-known rules of base pairing (Watson andCrick), where adenine (A) pairs with thymine (T) or uracil (U); andwhere guanine (G) pairs with cytosine (C), wherein correspondingbase-pairs are part of complementary, anti-parallel nucleotide strands.The base pairing results in a specific hybridization betweenpredetermined and complementary nucleotides. The base pairing is thebasis by which enzymes are able to catalyze the synthesis of anoligonucleotide complementary to the template oligonucleotide. In thissynthesis, building blocks (normally the triphosphates of ribo ordeoxyribo derivatives of A, T, U, C, or G) are directed by a templateoligonucleotide to form a complementary oligonucleotide with thecorrect, complementary sequence. The recognition of an oligonucleotidesequence by its complementary sequence is mediated by corresponding andinteracting bases forming base pairs. In nature, the specificinteractions leading to base pairing are governed by the size of thebases and the pattern of hydrogen bond donors and acceptors of thebases. A large purine base (A or G) pairs with a small pyrimidine base(T, U or C). Additionally, base pair recognition between bases isinfluenced by hydrogen bonds formed between the bases. In the geometryof the Watson-Crick base pair, a six membered ring (a pyrimidine innatural oligonucleotides) is juxtaposed to a ring system composed of afused, six membered ring and a five membered ring (a purine in naturaloligonucleotides), with a middle hydrogen bond linking two ring atoms,and hydrogen bonds on either side joining functional groups appended toeach of the rings, with donor groups paired with acceptor groups.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.alpha-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes e.g. so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues preferably joinedexclusively by peptide bonds, whether produced naturally orsynthetically. A polypeptide produced by expression of a non-host DNAmolecule is a “heterologous” peptide or polypeptide. The term“polypeptide” as used herein covers proteins, peptides and polypeptides,wherein said proteins, peptides or polypeptides may or may not have beenpost-translationally modified. Post-translational modification may forexample be phosphorylation, methylation and glycosylation.

The term “expression” refers to the biosynthesis of a gene or a geneproduct.

To “hybridize” means annealing nucleic acid strands from differentsources; that is, to form base pairs between complementary regions oftwo strands of DNA that were not originally paired. The term“hybridization under stringent conditions” is defined according toSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor, Laboratory Press (1989), 1.101-1.104. Preferably, hybridizationunder stringent conditions means that after washing for 1 h with 1 timesSSC and 0.1% SDS at 50 degree C., preferably at 55 degree C., morepreferably at 62 degree C. and most preferably at 68 degree C.,particularly for 1 h in 0.2 times SSC and 0.1% SDS at 50 degree C.,preferably at 55 degree C., more preferably at 62 degree C. and mostpreferably at 68 degree C., a positive hybridization sitmal is observed.

A stretch of “Complete homology” is defined as a match of pairingnucleotides along the sequence of the interacting nucleotides; innatural occurring RNA the pairing of A with U and G with C.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. If a promoter is an inducible promoter, then therate of transcription increases in response to an inducing agent. Incontrast, the rate of transcription is not regulated by an inducingagent if the promoter is a constitutive promoter. Repressible promotersare also known.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a promoter. For example, a regulatory element may contain anucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineor ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter. Simplervectors called “transcription vectors” are only capable of beingtranscribed but not translated: they can be replicated in a target cellbut not expressed, unlike expression vectors. Transcription vectors areused to amplify their insert.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector.

Transfection describes the introduction of foreign material intoeukaryotic cells. The term ‘transfection’ for non-viral methods is mostoften used in reference to mammalian cells, while the term‘transformation’ is preferred to describe non-viral DNA transfer inbacteria and non-animal eukaryotic cells such as fungi, algae andplants. Both chemical and physical methods may be employed to transfectcells.

A “polypeptide” is a polymer of amino acid residues preferably joinedexclusively by peptide bonds, whether produced naturally orsynthetically. A polypeptide produced by expression of a non-host DNAmolecule is a “heterologous” peptide or polypeptide. The term“polypeptide” as used herein covers proteins, peptides and polypeptides,wherein said proteins, peptides or polypeptides may or may not have beenpost-translationally modified. Post-translational modification may forexample be phosphorylation, methylation and glucosylation.

An “amino acid residue” can be a natural or non-natural amino acidresidue linked peptide bonds or bonds different from peptide bonds. Theamino acid residues can be in D-configuration or L-configuration. Anamino acid residue comprises an amino terminal part (NH₂) and a carboxyterminal part (COOH) separated by a central part comprising a carbonatom, or a chain of carbon atoms, at least one of which comprises atleast one side chain or functional group. NH₂ refers to the amino grouppresent at the amino terminal end of an amino acid or peptide, and COOHrefers to the carboxy group present at the carboxy terminal end of anamino acid or peptide. The generic term amino acid comprises bothnatural and non-natural amino acids. Natural amino acids of standardnomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adoptedin 37 C.F.R., section 1.822(b)(2) belong to the group of amino acidslisted in Table 1 herein below. Non-natural amino acids are those notlisted in Table 1. Examples of non-natural amino acids are those listede.g. in 37 C.F.R. section 1.822(b)(4), all of which are incorporatedherein by reference. Also, non-natural amino acid residues include, butare not limited to, modified amino acid residues, L-amino acid residues,and stereoisomers of D-amino acid residues.

TABLE 1 Natural amino acids and their respective codes. Symbols 1-Letter3-Letter Amino acid Y Tyr tyrosine G Gly glycine F Phe phenylalanine MMet methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucineT Thr threonine V Val valine P Pro proline K Lys lysine H His histidineQ Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine C Cys cysteine

An “equivalent amino acid residue” refers to an amino acid residuecapable of replacing another amino acid residue in a polypeptide withoutsubstantially altering the structure and/or functionality of thepolypeptide. Equivalent amino acids thus have similar properties such asbulkiness of the side-chain, side chain polarity (polar or non-polar),hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral orbasic) and side chain organization of carbon molecules(aromatic/aliphatic). As such, “equivalent amino acid residues” can beregarded as “conservative amino acid substitutions”.

The classification of equivalent amino acids refers in one embodiment tothe following classes: 1) HRK, 2) DENQ, 3) C, 4) STPAG, 5) MILV and 6)FYW

Within the meaning of the term “equivalent amino acid substitution” asapplied herein, one amino acid may be substituted for another, in oneembodiment, within the groups of amino acids indicated herein below:

-   i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His,    Asn, Gin, Ser, Thr, Tyr, and Cys,)-   ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu,    Ile, Phe, Trp, Pro, and Met)-   iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu,    Ile)-   iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)-   v) Amino acids having aromatic side chains (Phe, Tyr, Trp)-   vi) Amino acids having acidic side chains (Asp, Glu)-   vii) Amino acids having basic side chains (Lys, Arg, His)-   viii) Amino acids having amide side chains (Asn, Gln)-   ix) Amino acids having hydroxy side chains (Ser, Thr)-   x) Amino acids having sulphor-containing side chains (Cys, Met),-   xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,    Thr)-   xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and-   xiii) Hydrophobic amino acids (Leu, Ile, Val)

The present invention also relates to variants of Hsp70, or fragmentsthereof, wherein the substitutions have been designed by computationalanalysis that uses sequence homology to predict whether a substitutionaffects protein function (e.g. Pauline C. Ng and Steven Henikoff, GenomeResearch, Vol. 11, Issue 5, 863-874, May 2001).

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to +/−20%, such as +/−10%,for example +/−5%.

DESCRIPTION OF THE DRAWINGS

FIG. 1

The effects of Hsp70 on aSMase binding to BMP and ceramide levels. (A)Binding of 0.2 μM aSMase to BMP-containing liposomes at pH 4.5 as afunction of pre-bound Hsp70 (experiment analogous to Example 1, seematerials and methods herein for further details). Hsp70 was allowed todissociate for 10 min, hereby reaching a lower asymptote fordissociation before addition of aSMase). (B) Confocal microscopy andquantification of ceramide levels in wild-type (WT) and Hsp70-transgenic(Hsp70-TG) iMEFs. Immunodetection was performed with a mouse monoclonalantibody against ceramide (clone 15b4). Quantification was done based onlaser scanning micrographs from 6 predefined areas, after whichquantification was done in LSM Duo software.

FIG. 2

The effect of rHsp70 on acid sphingomyelinase activity in iMEF-WT(immortalized murine embryonic fibroblasts, wild type). rHsp70 wasadministered to cells at 3 nM, 30 nM and 300 nM, and the activity ofaSMase measured (A500 is a measure of produced ceramide that increasesthe turbidity). Control cells were treated with BSA (bovine serumalbumin).

FIG. 3

Acidic SMase activity in different fibroblasts. NPDA: Niemann-Pickdisease type A.

FIG. 4

Scheme of major sphingolipid hydrolysis. Exohydrolytic breakdown ofsphingolipids with short hydrophilic headgroups requires non-enzymaticco-factors, sphingolipid activator proteins (SAPS or saponins). Both,inherited deficiencies of the respective enzyme as well as of thecorresponding activator protein causes lysosomal lipid storage andresults in the expression of various sphingolipidoses. From Ferlintz etal., Chem. Phys. Lipids, (102) 35-43, 1999.

FIG. 5

Lysosomal Hsp70 stabilizes lysosomal membranes. (a) Representativeconfocal images of U-2-OS cells incubated with 300 nM rHsp70-AF488(green) for 24 h, fixed and stained for lysosomal integral membraneprotein-1 (LIMP-1; red). For co-localization with other organellemarkers see FIG. 9. (b) U-2-OS cells were incubated with 300 nMrHsp70-AF488 for 24 h before quantification of rHsp70-AF488 in membranes(memb.) and supernatant (sup.) obtained by repeated freeze/thaw cyclesand centrifugation the light membrane fraction (LMF). The immunoblotanalyses of lysosome-associated membrane protein 2 (LAMP-2) andcathepsin B (Cat B) demonstrate the validity of the fractionationprocedure. (c) Representative still images of U-2-OS cells exposed tophoto-oxidation (acridine orange and blue light). The loss of lysosomalintegrity is visualized by the loss of red and increase in greenstaining. (d and e) U-2-OS cells were incubated with indicatedrecombinant proteins (300 nM) for 24 h, and analyzed for lysosomalintegrity upon photo-oxidation. When indicated, cells were treated withindicated siRNAs for 48 h prior to the addition of recombinant proteins(e). The values represent means±SD for three (d) or five (e) independentexperiments. Representative immunoblots of indicated proteins fromU-2-OS cells left untreated or treated with control or Hsp70 siRNAs areshown on the right. Scale bars: 20 μm (a and c).

FIG. 6

A pH-dependent interaction between Hsp70 and BMP stabilizes lysosomalmembranes. (a) Relative changes in liposome 90° light scattering uponaddition of rHsp70 (in 0.12 nmol aliquots) to liposomes containingindicated lipids (χ=0.2) at pH 7.4 (left) and pH 6.0 (right). (b) U-2-OScells were left untreated (−) or incubated with 50 μg/ml anti-BMP orcontrol IgG for 7 h before addition of vehicle (−) or 300 nM rHsp70 for24 h, and analyzed for lysosomal integrity upon photo-oxidation. (c)U-2-OS cells were left untreated or incubated with 50 μg/ml anti-BMP orcontrol IgG for 7 h before addition of vehicle (−) or 25 μM cisplatinfor 24 h and analyzed for apoptotic cell morphology following Hoechst33342 staining. (d) Interaction of rHsp70 and its mutants with POPC/BMP(χBMP=0.2) liposomes at pH 6.0 as measured by changes in relative peakfluorescence intensity. Protein concentrations were 0.36 μM (rHsp70),0.5 μM (□ATP) and 0.35 μM (□PBD) (left) or 0.43 μM (right), andliposomes were added in 10 μM aliquots. (e) BIAcore analysis ofinteractions between wild type rHsp70 (WT) and its deletion (□ATP and□PBD) and point (W90F and W580F) mutants with immobilized LUVs at pH 4.5(average diameter: 100 nm; total lipid concentration: 0.1 mM;composition: sphingomyelin (χ=0.1), phosphatidylcholine (χ=0.5),cholesterol (χ=0.2) and BMP (χ=0.2)). Liposomes were injected untilequilibrium (100 s), and indicated concentrations (left) or 300 nM(right) of recombinant proteins in sodium acetate buffer (50 mM, pH 4.5)were injected for 200 s at a flow rate of 20 μl/min followed by adissociation phase for 10 min. □RU is defined as the difference betweenthe response signal measured after liposome equilibrium andprotein-liposome equilibrium. (f and g) U-2-OS cells were left untreated(Control) or incubated with indicated recombinant Hsp70 proteins (300nM) for 24 h, and analyzed for lysosomal integrity upon photo-oxidation(f), or treated with vehicle (white bars) or 25 μM cisplatin (Blackbars) for 24 h and analyzed for the apoptosis like morphology (g). (h)Ribbon and Molecular surface models of the ATPase domain of Hsp70. ATP(van der Waal-surface representation) can be visualized bound in theATP-binding pocket. Green and purple spheres denote the van derWaals-surface of the coordinated Calcium and Sodium ions, respectively.Notice the positively charged part of the domain in the bottom and theposition of W90.

The values represent means±SD for a minimum of five independentexperiments (b, c, f and g).

FIG. 7

Hsp70 stimulates ASM activity that in turn stabilizes lysosomes. (a)Biacore measurement of binding of 200 nM rASM to BMP-containingliposomes at pH 4.5 as a function of pre-bound rHsp70. The experimentswere performed as described in the legend for FIG. 6e with rASM addedfor 180 sec after the 10 min rHsp70-dissociation phase followed by yet a10 min dissociation phase. (b) ASM activity in the lysates of wild-type(WT) and Hsp70 transgenic (Hsp70) MEFs (left panel) and in WT MEFsincubated with 300 nM rHsp70 for 24 or 48 h as indicated. (c and d)Viability (MTT reduction; c) and cytosolic cathepsin activity (zFRase;d) in WT and Hsp70 iMEFs treated with indicated concentrations ofdesipramine for 3 h. (e) Live single-cell imaging of loss of lysosomalintegrity (photo-oxidation in WT and Hsp70 MEFs as well as Hsp70 MEFsincubated for 3 h with 12.5 and 25 μM Desipramine (left and rightpanels, respectively). Loss of red (left panel) and increase in greenfluorescence (right panel) was continuously measured to give fullkinetic curves for the loss of lysosomal integrity. 25-60 cells wereexamined pr. experiment from pre-defined areas), p<0.001 for Hsp70 vs.WT and Hsp70+despramine vs. Hsp70. All values represent means±SD for aminimum of 3 independent experiments.

FIG. 8

rHsp70 stimulates ASM activity, stabilizes lysosomes and decreases thelysosomal volume in NPDA fibroblasts. (a) Live single-cell imaging oflysosomal stability of primary fibroblasts from a patient with NPDAanalyzed as in FIG. 3e , p<0.001. (b) ASM activity of NPDA fibroblastsleft untreated or treated with 300 nM rHsp70 for 48 h (left panel), orwith 150 nM rASM alone or in combination with 300 nM rHsp70 for 24 h(right panel). The p values were calculated from the obtained enzymaticvelocity (DA500/mg protein/min). The picture on the right demonstratesthe endocytic uptake of rASM (green) and its localization to thelysosomal compartment as visualized by co-staining with Lysotracker Red.(c) Lysosomal stability of NPDA fibroblasts left untreated or treatedfor 24 h with 300 nM rHsp70, 150 nM aSMase or a combination of rHsp70and aSMase was analyzed as in FIG. 3e . p<0.001 for all treatments ascompared with untreated cells. (d) Quantification of lysosomal area ofconfocal cross sections of cells in NPDA fibroblasts left untreated ortreated for 24 h with 300 nM BSA, 300 nM rHsp70, 150 nM rASM (150 nM) ora combination of rHsp70 and rASM. The picture on the right demonstratesthe effect of rHsp70 (green) on the volume of the lysosomal compartment(red) in NPDA fibroblasts. White arrows indicate cells with endocytosedrHsp70 and diminished lysosomal compartment. The values representmeans±SD for 3 independent experiments. Scale bars=20 μM. UT=untreated.

FIG. 9

Colocalization of endocytosed rHsp70-AF488 with lysosomes.Representative confocal images of U-2-OS cells incubated with 300 nMrHsp70-AF488 (green) for 24 h, fixed and stained for the followingorganelle markers (red): lysosome-associated membrane-protein-1 (LAMP-1;lysosomes), LAMP-2 (lysosomes), LBPA/BMP (6C4; endo-lysosomalcompartment), cut c (mitochondria), SERCA (ER) and golgin-97 (Golgi).Scale bars: 20 μm (LAMP-1, LAMP-2 and BMP) or 10 μm (Cyt c, SERCA andGolgin-97).

FIG. 10

Interaction of rASM (recombinant aSMase) and BMP in the presence ofrHsp70. (a) interaction of rASM with immobilized anionic liposomes(average diameter is 100 nm, total lipid concentration is 0.1 mM, andcomposition; 10 mol % sphingomyelin, 50 mol % phosphatidylcholine, 20mol % cholesterol and 20 mol % BMP) at pH 4.5. Response signals measuredsubsequent to the binding of the liposomes where defined as zero. (b)The effect of prebound rHsp70 on subsequent binding of rASM. Indicatedamounts of rHsp70 were incubated with immobilized anionic liposomesidentically to (a). After a 10 min dissociation phase of rHsp70, 200 nMrASM was added for 180 s followed by 10 min dissociation.

FIG. 11

Effect of the small molecule Hsp70 inducer; Benyl Alcohol onNiemann-Pick Type A (NPDA) patient fibroblasts. (A) Induction of Hsp70in NPDA Götz by Benzyl alcohol in a dose-dependent manner (proteinexpression). (B) Increased stability of NPDA Götz lysosomes aftertreatment of NPDA Götz cells with 40 mM Benzyl Alcohol. (C) Decreasedpathology in NPDA Götz cells after treatment with 40 mM Benzyl Alcohol,as measured by lysosomal cross-sectional area (method further detailedin Example 2).

FIG. 12

Effect of aSMase depletion on lysosomal stability. Small interferingRNAs (siRNAs) targeting acid Sphingomyelinase (si938, si1257, si1340)and a control siRNA (mm) were transfected into U2OS cells usingOligofectamine (Invitrogen) according to the manufacturers guidelines.Concentration of siRNAs: 50 nM. After 72 h hours knockdown was confirmedvia RT-PCR (not shown) and cells analyzed for lysosomal stability vialive single-cell imaging of acridine orange mediated photooxidation.Increase in green fluorescence was continuously measured to give fullkinetic curves for the loss of lysosomal integrity. As evident form thegraphs cells treated with siRNAs targeting aSMase show a marked decreasein lysosomal stability. The method is further explained in Example 2.

FIG. 13

Treatment of all NPDA/B cell lines with rHsp70 dramatically reverses thelysosomal pathology, i.e. reduces the cross-sectional area of lysosomes.Quantification of lysosomal area of confocal cross sections of cells ofNiemann-Pick Disease Type A and B fibroblasts (NPDA/NPDB) and normalfibroblasts (BJ) left untreated or treated for 24 h with 300 nM BSA orDextran as control, or treated for 24 h with 300 nM rHsp70, 150 nMrhaSMase or a combination thereof. NPDA cells treated for 24 h with 300nM rHsp70-W90F (W90F)—the Hsp70-mutant which does not interact with BMP,has an effect comparable to control cells. See Example 2 for methods.

FIG. 14

Increased activity, of aSMase in Hsp 70 transgenic fibroblasts andrHsp70 treated NPDA fibroblasts. Mass spectroscopic analysis of lipidspecies (sphingomyelin and ceramide as indicated) in immortalized mouseembryonic fibroblasts (iMEFs), either wild type (WT) or Hsp70-transgenic(TG) (A and B), as well as Niemann-Pick Disease type A patientfibroblasts (NPDA 83/24) either left untreated or treated with rHsp70(C). The lower levels of sphingomyelin and higher levels of ceramideindicate an increased activity of acidic sphingomyelinase.

FIG. 15

Reversion of pathology in Farber Disease Patient Fibroblasts.Quantification of lysosomal area of confocal cross sections of cellsfrom Farber Disease Patients. Farber Disease patient fibroblasts (Farber89/73 and Farber 89/78) were left untreated or treated for 24 h with 300nM BSA or 300 nM rHsp70 as indicated. As evident from the figures, thetreatment of Farber disease patient fibroblasts with rHsp70 dramaticallyreverses the lysosomal pathology, i.e. reduces the cross-sectional areaof lysosomes. See Example 2 for a description of methods.

FIG. 16

Hsp70 increases endocytic uptake of other molecules. Panel A:immortalized mouse embryonic fibroblasts (iMEF), either wildtype (WT) ortransgenic for Hsp70 (TG) where incubated with 20 μg/mL AlexaFluor-488-labelled BSA (BSA*) for 24 h. Endocytic uptake was verified byfluorescence microscopy (not shown) (see example 2). Cells where thenharvested and analyzed for uptake of BSA*. As evident from the figurethe Hsp70-transgenic iMEFs had a significantly higher uptake of BSA*than wildtype iMEFs. Panel B: U2OS osteosarcoma cells where incubatedwith 20 μg/mL BSA* for 24 h either with 3000 nM rHsp70 or without asindicated. Endocytic uptake was verified by fluorescence microscopy (notshown) (see example 2). Cells where then harvested and analyzed foruptake of BSA*. As evident from the figure, the U2OS cells in which BSA*and rHsp70 where added together had a significantly higher uptake ofBSA* than cells incubated with BSA* alone.

DETAILED DESCRIPTION OF THE INVENTION

As is demonstrated by the present inventors, Hsp70 exerts a major partof its cytoprotective effect through a direct interaction withendo-lysosomal membranes; an interaction which is orchestrated by aspecific phospholipid, namely BMP (bis(monoacylglycero)phosphate). BMPis present only in late endosomes and lysosomes. The inventors show thatthe Hsp70-BMP interaction is dependent on the N-terminal ATP-ase domainof Hsp70, specifically tryptophan 90, and also that the interaction ispH-dependent. The interaction between Hsp70 and BMP is essential for themembrane-stabilizing effect of Hsp70, by providing a platform formodulating the stability of a specific subset of lysosomal enzymes, andpreventing destabilization of lysosomal membranes with ensuing releaseof lysosomal enzymes. These findings form the basis for a new andpromising treatment modality for the lysosomal storage disorders, asdisclosed herein.

Lysosomes

Since the discovery of lysosomes by de Duve in 1955, the view of thisorganelle has been dominated by the dogma that it is solely the terminusof the endocytic pathway in animal cells—a compartment housing a vastarray of hydrolases, that, if released into the cytosol, cause necrosisand tissue inflammation. This view of the lysosomes as, at best, agarbage disposal unit, and at worst, an unspecific “suicide bag” haschanged dramatically due to recent discoveries that provide evidence fornumerous more specific tasks for lysosomes and their contents.

Lysosomal Hydrolases

As the main compartment for intracellular degradation and subsequentrecycling of cellular constituents, the lysosomes receive both hetero-and autophagic cargo, which in the lumen of this organelle find theirfinal destination. The degradation is carried out by a number of acidhydrolases (phosphatases, nucleases, glycosidases, proteases,peptidases, sulfatases, lipases, etc.) capable of digesting all majorcellular macromolecules. Among the best-studied lysosomal proteases isthe family of cathepsin proteases. The cathepsins can be divided intothree sub-groups according to their active site amino acid, i.e.cysteine (B, C, H, F, K, L, O, S, V, W and X/Z), aspartate (D and E) andserine (G) cathepsins. The cathepsins function optimally at the acidicpH of the lysosomes (pH 4-5) although they can still function at theneutral pH outside the lysosomes, albeit having decreased stabilityand/or altered specificity.

Until recently the function of cathepsins was thought to be limited tointralysosomal protein-turnover, and the degradation of theextracellular matrix once secreted. However, during the past few yearsmany of the cathepsins have been accredited with more specific functionsincluding roles in bone remodeling, antigen presentation, epidermalhomeostasis, prohormone processing, protection of cytotoxic lymphocytesfrom self-destruction after degranulation, maintenance of the centralnervous system in mice, angiogenesis, cancer cell invasion as well asprogrammed cell death (PCD).

Apart from the breakdown of proteins, the lysosomes and late endosomesare also responsible for the metabolism of cellular lipids, such as theglycosphingolipids, through a series of endolysomal enzymes andco-ensymes, whose proper function depend on the lipid composition of theintra-lysosomal membranes. The importance of functional endolysosomallipid metabolism can be easily appreciated by the fact that clinicaldisease is apparent in case of dysfunction at any stage of sphingolipidmetabolism, giving rise to diseases such as Tay-Sachs, Sandhoff, Farber,Fabry, Gaucher, Krabbe and Niemann-Pick disease.

Trafficking to and from the Lysosomes

The traffic of endocytic membranes serves an essential role in themammalian cell through its delivery of membrane components, varioussolute molecules and receptor-associated ligands to a range ofintracellular compartments. Whilst the various endocytic routes untilrecently appeared simple, with the main pathways converging on thelysosomes, where degradation and possible recycling back to the plasmamembrane would take place, recent evidence shows that these pathways aremore complex than first imagined.

The endocytic route Endocytosis is best understood in terms of thereceptor-mediated endocytosis of molecules via the formation ofclathrin-coated pits, although a variety of non-clathrin mediatedendocytic routes (e.g. macropinocytosis, phagocytosis, uptake viacaveolae-formation and non-clathrin-coated-pit formation) have also beenidentified. The nomenclature of the endocytic system has not been fullystandardized, and the commonly used term “early endosome” actuallydescribes two distinct endosomal compartments—the sorting endosome andthe endocytic recycling compartment (ERC). In the conventionalreceptor-mediated endocytic pathway, receptors such as the transferrinreceptor, the low density lipoprotein receptor and the mannose6-phosphate receptor (MPR) concentrate into clathrin-coated pits on thesurface of the plasma membrane by virtue of interactions betweensequence motifs in their cytoplasmic tails and elements in the clathrincoat. After shedding of its clathrin-coat, the newly formed endosomefuses with other endosomes and pre-existing sorting endosomes to becomea sorting endosome. As the name implies, its primary task is to sortnewly acquired components to their correct locations. The three knowndestinations include the plasma membrane, the late endosomes and theERC. As the sorting endosome matures, it experiences a drop in pH, whichfacilitates the release of receptor-bound ligands into the lumen of theendosome. Before the full maturation of the sorting endosome into thelate endosome, however, the molecules destined to recycling must besorted out. It is believed that this process takes place through thepinching off of narrow tubules, a process, which favors the sorting ofmembrane proteins from solute molecules as the surface-area-to-volumeratio of the tubules is greater than that of the vesicular sortingendosome. The pinched-off-tubules can either relay the membrane proteinsdirectly back to the plasma membrane (the direct return pathway) or tothe ERC. The ERC is mainly a collection of tubular organelles, whoselocalization varies between cell types. While the ERC is capable ofsorting molecules to several different destinations, most of themolecules that transit via the ERC return to the plasma membrane.

As the sorting endosome matures, its luminal pH steadily drops, mainlydue to the action of the vacuolar-type proton ATPase (V-ATPase), whileshifts in membrane lipid and protein composition also occur. Themembrane traffic from the sorting endosome to the late endosome andfurther into the lysosome has been the scene of some controversy. Thedispute concerns whether this transport is best explained via vesiculartransport or by the maturation of the sorting endosome. Both modelsprovide for an intermediate between the sorting and the late endosome.While the maturation model argues that the vesicle, which reaches thelate endosome, is what remains after the removal of components from theformer sorting endosome, the pre-existing compartment model argues thattransport of molecules to the late endosomes occurs via an endocyticcarrier vesicle (ECV), a specific transport vesicle between pre-existingsorting and late endosomal compartments. Both the sorting and lateendosomal compartments are considered to be structurally more complexand to have more specialized functions than the carrier vesicles. Recentlive-cell imaging studies have reconciled mechanistic aspects of bothmodels, however, as vesicles arising from a dynamic early endosomenetwork can undergo a conversion in which they loose the small GTPaseRAB5 and recruit RAB7, a marker of late endosomes. Although theorganization of the endocytic pathway is functionally well defined, thenomenclature can be confusing. Functionally, the endocytic pathway isdefined by housekeeping receptors (e.g. the transferrin receptor) andother lipids and proteins being cycled through the earlyendosome/sorting endosome where receptor-ligand uncoupling occurs—butnot through late endosomes where proteolysis can occur. Beyond thesefunctional criteria however, the picture becomes cloudier when it comesto nomenclature, not least so as the generation of intraluminalvesicles, starting in the early endosomes and becoming more and moreprominent during the maturation to late endosomes, has given rise to theterm “multivesicular bodies” (MVB). This term has been usedinterchangeably as another name for the ECVs and late endosomes as wellas for all endocytic vesicles containing multivesicular regions orelements, including the hybrid organelle that forms when the lysosomesfuse with the late endosomes (which contain multivesicular structures).However, late endosomes contain more luminal membrane vesicles thanearly endosomes and are thus often the compartment described by the term“multivesicular bodies”.

Finally, a substantial amount of confusion in the field has arisen fromthe definition, or rather lack thereof, of late endosomes versuslysosomes. Both compartments are equally acidic and most, if not all,proteins present in lysosomes are also found in late endosomes.According to the maturation model, the late endosomes would beprecursors for the lysosomes, but given the gradual development, as thetheory suggests, a stringent classification could be very difficult toachieve. Recently, however, evidence has been presented for lysosomesand late endosomes being separate compartments, which then undergo both“kissing” events (transient fusions) as well as complete fusion events,after which the lysosomes can reform from the hybrid organelle.

The Biosynthetic Route

Apart from endocytosis, the late endosomes also receive cargo via theMPR pathway from the trans-golgi network (TGN) (the biosynthetic route).The cation-dependent MPR and the cation-independent MPR/Insulin-likegrowth factor-II (IGF-II) receptor share the task of delivery of newlysynthesized acid hydrolases from the TGN to the lysosomes. Therecognition of acid hydrolases by MPRs requires the addition ofcarbohydrates in the endoplasmic reticulum and the subsequentmodification and phosphorylation of the carbohydrate residues tomannose-6-phosphate moieties in the cis-Golgi The MPR-bound hydrolasesare first delivered to endosomes, where they dissociate from thereceptors due to the drop in the lumenal pH, hereby allowing thereceptors to recycle back to the TGN. The protein mainly responsible forthe sorting of the MPRs into clathrin-coated pits at the TGN, is anadaptor protein-1 (AP-1), although the Golgi-localized, γ-ear-containingADP ribosylation factor-binding proteins (GGAs) also play a part.Whether AP-1 and the GGAs work in concert or in fact target the two MPRsto different subcellular localizations is presently unknown. AP-1 ispart of an adaptor protein family consisting of four members, all ofwhich are heterotetrameric proteins utilized extensively in thesecretory and endocytic pathways. In addition to the above-mentionedrole of AP-1 in clathrin-coated pits formed in TGN, AP-1 and AP-2 areused in clathrin-coated pits during endocytosis at the plasma membrane,while AP-3 and AP-4 function in the trafficking of thelysosome-associated membrane proteins (LAMPs).

The Autophagic Route

Autophagy is the third well-characterized route by which macromoleculesreach the lysosome. Autophagy is an evolutionary conserved pathwayinvolved in the turnover of long-lived proteins and organelles. Itusually operates at low basal levels, although it can be induced, forexample under conditions of nutrient starvation. Under these conditionsmacroautophagy is the major pathway responsible for delivering materialto the lysosomes. Macroautophagy is characterized by a flat membranecistern wrapping around cytoplasmic organelles and/or a portion ofcytosol thereby forming a closed double-membrane bound vacuole, theautophagosome. The autophagosome finally fuses with lysosomes formingautophagolysosomes/autolysosomes, where the degradation and recycling ofthe engulfed macromolecules occur. The origin of the autophagosomemembrane is still not clarified. The endoplasmic reticulum, Golgi, aless-well characterized membrane compartment called the phagophore aswell as de novo synthesis have all been proposed as origins of theautophagosome membrane. Recent progress through yeast genetics and thesubsequent discovery of mammalian homologues is rapidly enhancing theunderstanding of the process of autophagy and will hopefully shed lightalso on the origin of the autophagosomal membrane in the near future.

There are also other routes by which the lysosomes receive autophagiccargo. A rather indiscriminate process termed microautophagy ischaracterized by engulfment of cytosol by the lysosomes throughinvaginations of the lysosomal membrane. Besides the macromolecules,which are present in the engulfed cytosol, this process may also involvethe uptake of organelles such as peroxisomes. Finally,chaperone-mediated transport of cytosolic proteins into the lysosomallumen presents a more direct and selective form of autophagy. Thispathway is dependent on the presence of the constitutively expressedmember of the Heat shock protein 70 family, Hsc70, on both sides of thelysosomal membrane. The process is furthermore dependent on therecognition of a KDEL sequence motif in target proteins by LAMP-2a.

Reformation of Lysosomes and Lysosomal Secretion

After fusion of lysosomes with late endosomes or autophagosomes, thelysosomes are reformed from the resultant hybrid organelles throughsequestration of membrane proteins and condensation of the lumenalcontent. Of the membrane proteins that need to be removed or recycledfrom the hybrid organelle, the most obvious are the MPRs, as they bydefinition are absent from lysosomes. The lysosomes, however, cannot beseen as the terminal point of the endocytic pathways as they are alsoable to form secretory lysosomes through fusion with secretory granules,a process that is Ca²⁺-dependent and was first recognised in secretorycells of haematopoietic origin. However, evidence also exists for aCa²⁺-regulated membrane-proximal lysosomal compartment responsible forexocytosis in non-secretory cells. The process of exocytosis isdependent on the protein Rab27a, a member of the Rab protein family,which counts more than 60 members. The Rabs are small GTPases that havekey regulatory roles in most membrane-transport steps including vesicleformation, motility, docking and fusion. At least 13 Rab proteins areutilised in the endocytic pathways in order to determine the fate of thevarious endocytosed molecules and their vesicles.

Programmed Cell Death

Regulation of overall cell number as well as the amount of cellsconstituting the different tissues along with the need for a mechanismof eliminating unwanted cells is of fundamental importance inmulticellular organisms. Programmed cell death is the means to this end,endowing the multicellular organism with the potential to rid itself ofunwanted cells without the leakage of cellular constituents, thusavoiding the inflammation associated with necrosis, the conceptualcounterpart to programmed cell death.

Apoptosis

The word apoptosis is used in Greek to describe the “dropping off” or“falling off” of petals from flowers, or leaves from trees and was firstcoined by Currie and colleagues in 1972 to describe a common type ofprogrammed cell death, which the authors had observed in a number oftissues and cell types. The authors had noticed that the events theyobserved had significant morphological similarities, which were distinctfrom the morphological features characterizing cells undergoingpathological, necrotic death and suggested that these commonmorphological features might be due to an identical underlying process.

When cells die by apoptosis, they undergo a series of transformingevents. Amongst these events, and essential for the characteristicapoptotic phenotype, is the activation of caspases—a family of cysteineendopeptidases, which cleave substrates at specific aspartate residues,hence the name. The activation of the caspases lead to proteolyticprocessing of other caspases as well as a host of other changes in theoverall protein activities within the cells, ultimately producing thecharacteristic morphological features associated with thecaspase-activation and thus, per definition, apoptosis. The classicalapoptotic features include cell shrinkage and blebbing of thecytoplasmic membrane, condensation of chromatin within the nucleus inclear, geometrical shapes, fragmentation of DNA into ˜200 bp integers,the so-called nucleosomal ladder, cellular detachment from itsneighboring cells and disintegration of the cell into small, enclosedvesicles termed apoptotic bodies. In a multicellular environment theseapoptotic bodies are ultimately phagocytosed by macrophages orneighboring cells hereby completing the removal of the unwanted cell.

Programmed Cell Death

Programmed cell death (PCD) is not synonymous with apoptosis althoughone could be inclined to think so based on the amount of literatureusing these terms indiscriminately. The term PCD is gradually takingover, but the term apoptosis is still used to describe a cell deathprogram orchestrated by the activation of caspases, in particularcaspase-3. However, the ability of certain cells to survive theactivation of pro-apoptotic caspases as well as PCD with completeabsence of caspase activation and caspase-activation leading tonon-apoptotic PCD, has revealed a remarkable plasticity of the cellulardeath programme(s) and PCD can thus be more accurately defined as celldeath dependent on signals or activities within the dying cell. It hasbeen suggested that PCD can be subdivided into apoptosis, apoptosis-likeand necrosis-like PCD, according to the nuclear morphology of the dyingcells, each definition coined to distinct morphological characteristics,the main feature being the shape of chromatin condensation or theabsence hereof, although it would be preferable to make distinctions ofPCD based on the signaling pathways participating under any given set ofconditions leading to PCD. This way of distinguishing between differentmodes of PCD is not yet applicable however, as the threads leading tothe varying kinds of cell death remains to be sorted out.

Necrosis

Necrosis is the conceptual counterpart to PCD, as it cannot be preventedby any other means than removing the stimulus giving rise to thenecrosis. This mode of cell death is usually seen during pathologicalinsults to an organism.

The Molecular Machinery of Programmed Cell Death

Apoptosis

As mentioned in the previous section, apoptosis is defined by theactivation of members of the family of cysteine endopeptidases known asthe caspases and the morphology associated with their activation. Thecaspases reside in cells as inactive zymogens, which can be rapidlyactivated by proteolytic processing. This processing proceeds in ahierarchic cascade in which an apoptotic stimulus activates an initiatorcaspase (e.g. caspase-8 and -9), which in turn activates the next levelin the hierarchy, the effector caspases (e.g. caspase-3, -6 and -7). Thelatter are considered the executioners of apoptosis as they cleave anumber of substrates, the processing of which ultimately leads to thephenotype associated with apoptosis. The apoptotic programme can beactivated by a variety of stimuli, which can be broadly divided intoextracellular and intracellular stimuli, the latter seeing themitochondrion as an essential player. The extracellular stimuli and thefollowing response giving rise to apoptosis are also referred to as theextrinsic signaling pathway and are comprised of a series of eventsstarting with activation of one of a variety of death receptors such asFas/Apo-1/CD95, TNFR or TRAIL. Upon binding of their appropriate ligand,these receptors recruit death domain (DD)-containing adaptor molecules,such as TRADD (TNFR1-associated death domain protein) and FADD(Fas-associating protein with death domain), through interaction withthe DD present in the receptors. These adaptor molecules then recruitcaspase-8 to the receptor complex, where the caspase is activated,possibly by proximity-induced autocatalytic processing. In certain cells(the so-called type I cells) caspase-8 then directly cleaves andactivates procaspase-3, whereas in type II cells, the substratum forcaspase-8 is the cytoplasmic protein Bid. The cleavage of Bid generatesa fragment (truncated Bid (tBid)), which induces the oligomerisation,translocation and insertion of two pro-apoptotic Bcl-2 family members,Bax and Bak into the outer mitochondrial membrane. This insertionmediates the release of the electron-carrier cytochrome c (CytC) fromthe mitochondrial intermembrane space along with a host of otherproteins, the most prominent of which include Apoptosis Inducing Factor(AIF), Smac/DIABLO which antagonizes the effects of the proteins knownas inhibitors-of-apoptosis (IAP) proteins and endonuclease G, a DNAse.It should be noted, that although this is the pivotal point in thetheories of caspase activation through mitochondria, no conclusiveevidence has been presented with regard to how the insertion of Bax andBak facilitates the release of cytochrome c. Upon release from themitochondrion, CytC accumulates in the cytoplasm, where it binds to theprotein Apaf-1 (apoptotic protease-activating factor-1) resulting in aconformational change, which promote oligomerisation of Apaf-1. Thisoligomer then binds procaspase-9 through homotypic interactions betweencaspase recruitment domains (CARDs) resulting in the formation of acomplex called the apoptosome. The formation of this complex leads to agreatly enhanced enzymatic activity of pro-caspase-9, the activity ofwhich leads to the proteolytic activation of caspase-3.

Apoptosis can also be triggered by intracellular factors elicitingmitochondrial outer membrane permeabilisation (MOMP), a process known asthe intrinsic pathway. These factors include second messengersassociated with cellular stress such as Ca²⁺, NO and arachidonic acid aswell as bilirubin, bile salts and stimuli which can give rise to proteindenaturation and nuclear and mitochondrial DNA damage such as ionizingradiation, heat stress, reactive oxygen species (ROS) andchemotherapeutic agents. In the event of nuclear DNA damage, this issensed by a variety of protein kinases, which depends on the form of DNAdamage but also the noxa eliciting it. The activity of these kinasesinduce the accumulation of p53, which can then act as a transcriptionfactor, giving rise to an enhanced transcription of pro-apoptotic genessuch as Bax, Noxa and PUMA, all of which can induce MOMP. At themitochondrial level, p53 induces the expression of mitochondrial enzymesthat locally generate ROS as well as a mitochondrial matrix protein(p53AIP1), which overexpression triggers loss of mitochondrial membranepotential and apoptosis.

The induction of MOMP by p53 or by the action of the intrinsic stimulidescribed above is the point at which the intrinsic and extrinsicpathways converge, the route of the intrinsic pathway following the onealready described for the extrinsic with release of cytochrome c,formation of the apoptosome and activation of caspase-3 constituting thefinal steps towards the demise of the unwanted cell.

The Alternatives to Apoptosis Within the past decade, the exclusive roleof caspases as the executioners of PCD has been challenged and mountingevidence suggest that there is more to life—and especially death—of acell than can be ascribed to the caspases alone.

As newly developed caspase-specific pharmacological inhibitors as wellas inactivation of caspase-pathways by factors such as energy depletion,nitrative/oxidative stress and members of the inhibitor of apoptosisprotein (IAP) family did not always stop the progression towards death,they revealed, or even enhanced, a subset of underlyingcaspase-independent death-programs. These programs includedeath-receptor initiated pathways as well as pathways elicited by cancerdrugs, growth-factor deprivation, staurosporine, Bax-related proteinsand the depletion of Hsp70. The morphological features of thesecaspase-independent death programs are often reminiscent of the onesobserved for classical apoptosis, and experimental support for a rolefor other proteases such as cathepsins, calpains and serine proteases asessential cofactors either upstream or downstream of caspases wasrapidly growing. The argument is strengthened by the findings that manynon-caspase proteases are able to cleave at least some of the classiccaspase substrates, which might explain some of the similaritiesobserved between the caspase-dependent and -independent deathprogrammes.

Although one can argue the relevance of such death programmes, as theyare masked by the efficacy of the caspases, evidence is gathering for anevolutionarily conserved role for lysosomal cathepsin proteases in celldeath programs initiated as a response to various stimuli such as deathreceptors of the tumor necrosis factor receptor family, hypoxia,oxidative stress, osmotic stress, heat and anti-cancer drugs.

Lysosomal Involvement in Programmed Cell Death

While the role of lysosomes and their hydrolases in the clean-up phaseof PCD, i.e. the engulfment of apoptotic cells and bodies by neighboringcells or phagocytes, is well established, it has taken a long time torecognize the importance of lysosomes and lysosomal hydrolases in themore immediate events of PCD. One of the reasons for this delay may bethe fact that the methyl ketone peptide inhibitors commonly used toassess the role of caspases in PCD (e.g. zVAD-fmk, Ac-DEVD-fmk,Boc-D-fmk, etc) also inhibit other cysteine proteases, including severalcysteine cathepsins. Even nine years after the recognition of thiscross-reaction, protective effects with these inhibitors atconcentrations capable of inhibiting non-caspase proteases are stilloften interpreted as a proof for caspase-mediated death pathways, andthe role of other cysteine proteases in PCD thus continues to beunderestimated. The discovery of lysosomal PCD may have beenadditionally delayed, because the lysosomal ultrastructure appearsintact in apoptotic cells analysed by electron microscopy. Thus, thelysosomal rupture has until recently been considered as anall-or-nothing switch during late stages of uncontrolled necrotic celldeath and tissue autolysis. However, new techniques allowing a moreprecise assessment of the lysosomal membrane integrity have revealedthat lysosomes with normal ultrastructure may have leaked part of theirenzymes, and that partial lysosomal membrane permeabilization (LMP) notonly occurs early in many death paradigms, but can in fact triggerapoptosis and apoptosis-like PCD.

Lysosomal membrane permeabilizaion (LMP) and its consequences Studieswith various compounds that directly target the integrity of thelysosomal membranes, such as H₂O₂, L-leucyl-L-leucine methyl ester,osmotic stress, sphingosine, the lysosomotropic antibiotics norfloxacinand ciprofloxacin and photo-oxidative lysosomal damage (photolysis),have convincingly proven that moderate lysosomal permeabilization canresult in PCD. A quantitative relationship between the amount oflysosomal rupture and the mode of cell death has been suggested toexplain the widely different morphological outcomes following LMP.According to this model, low stress intensities trigger a limitedrelease of lysosomal contents to the cytoplasm followed by apoptosis orapoptosis-like cell death, while high intensity stresses lead to ageneralized lysosomal rupture and rapid cellular necrosis. Accordingly,low concentrations of sphingosine, an acid ceramidase-generatedmetabolite of ceramide with detergent-like properties at low pH, inducespartial LMP and caspase-mediated apoptosis, whereas higherconcentrations result in massive LMP and caspase-independent necroticcell death. In this model, the death triggered by partial LMP can beinhibited by pharmacological inhibitors of cysteine and aspartatecathepsins, and the increase in the cytosolic cathepsin activityprecedes the activation of caspases and mitochondrial membrane potentialchanges suggesting a direct role for cytosolic cathepsins in the deathprocess. Importantly, the role of LMP and cathepsins in cell death isnot limited to the experimental models employing direct lysosomaldisrupters. LMP also participates in the execution of cell death inresponse to a wide variety of classic apoptotic stimuli, such asactivation of death receptors of tumour necrosis factor (TNF) receptorfamily, interleukin-1, p53 activation, growth factor starvation,microtubule stabilizing agents, etoposide, sigma-2 receptor activation,synthetic retinoid CD437, B cell receptor activation, staurosporine,osmotic stress, as well as small molecules identified in a screen fornovel cancer drugs that induce p53 independent apoptosis.

LMP as a Trigger of the Mitochondrial Apoptosis Pathway

The cytotoxic effects of LMP often rely, at least partially, on theactivation of the mitochondrial death pathway. An elegant microinjectionstudy has demonstrated that when localized to the cytosol, a singlelysosomal hydrolase, cathepsin D, is sufficient to trigger themitochondrial outer membrane permeabilization and apoptosis in humanfibroblasts at cellular doses corresponding to half of the totalcellular cathepsin D activity. Cathepsin D is, however, not sufficientto trigger PCD in all cell death models involving LMP. Otherwell-documented mediators of LMP-triggered PCD include cysteinecathepsins B and L as well as reactive oxygen species. It should,however, be emphasized that the role of other lysosomal hydrolases,lysosome-derived second messengers and LMP-induced acidification ofcytosol has not been appropriately ruled out. One of the links betweencathepsins and mitochondrial membrane permeabilization may be Bid, aproapoptotic BH3-only protein of the Bcl-2 family that can be processedand activated by several cysteine cathepsins, but not by cathepsin D, atcytosolic pH. Cathepsin D has, however, been suggested to cleave andactivate Bid in the acidic environment of the endolysosomal compartmentfollowing TNF receptor-1 (TNF-RI) internalization. According to thismodel, the endocytosis of the ligand-activated TNF-RI results in acidsphingomyelinase-mediated generation of ceramide, which then binds tothe inactive cathepsin D and activates it via autocatalytic processing.Cathepsin D may also activate Bax in a Bid-independent manner asdemonstrated in staurosporine-treated T cells. Also in fibroblaststreated with ciprofloxacine, LMP triggers mitochondrial membranepermeabilization through a Bid-independent activation of Bax and Bak. Inthis model system the Bax activation is independent of cathepsin D, butrelies instead on reactive oxygen species. It should be noted thatciprofloxacine-induced mitochondrial membrane permeabilization is notfully inhibited in cells lacking both Bax and Bak. The alternativemechanisms connecting LMP to the mitochondrial membrane permeabilizationmay include the direct effects of reactive oxygen species and/or lipidmediators such as arachidonic acid that can be generated in a cathepsinB-dependent manner.

Studies employing immortalized murine embryonic fibroblasts (MEFs) frommice deficient for individual cathepsins have clearly revealed thatdifferent cathepsins are engaged in the cell death execution dependingon the stimulus triggering LMP. Immortalized MEFs from cathepsin B and Ldeficient mice, but not from cathepsin D deficient mice, are highlyresistant to TNF, whereas the opposite picture emerges when the cellsare treated with staurosporine. Extensive studies on TNF-induced celldeath pathways have further revealed that the role of individualcathepsins in PCD depends on the cell type studied. As indicated above,TNF-induced death of immortalized MEFs depends on cysteine cathepsins,but not cathepsin D. Yet, cathepsin D depletion effectively protectsHeLa cervix cancer cells against TNF- and cisplatin-inducedcytotoxicity. This difference does not appear to be due to generaldifferences between human and murine cells, because cathepsin B alone ortogether with other cysteine cathepsins is also crucial for theeffective TNF-induced killing in human cervix (ME-180) and breast(MCF-7) cancer cell lines. The explanation for this diversity is as yetunknown, but varying expression levels of individual cathepsins andtheir inhibitors in different cell lines could play a role. Accordingly,the varying ability of different death stimuli to regulate theexpression levels of individual cathepsins or their inhibitors couldexplain the difference in response to different stimuli. For example,adriamycin and etoposide are known to enhance the expression ofcathepsin D via the activation of p53. Alternatively, other signalingpathways induced by various stimuli may co-operate with specificcathepsins.

Mitochondrion-Independent Death Pathways Induced by LMP

Importantly, the lethal effects of LMP and cytosolic cathepsins are notlimited to the activation of the intrinsic apoptosis pathway. In smallcell lung cancer cells treated with microtubule stabilizing drugs(paclitaxel, epothilone B and discodermolide), LMP occurs early in thedeath process and cysteine cathepsins mediate micronucleation and celldeath in a caspase-independent manner. In TNF-treated human carcinomacell lines LMP occurs downstream of mitochondrial outer membranepermeabilization. However, the inhibition of cysteine cathepsin activityor expression confers significant protection against TNF-induced celldeath without significantly inhibiting the effector caspase activation.Furthermore, cathepsin B is responsible for apoptosis-like changes, suchas chromatin condensation, phosphatidylserine exposure and plasmamembrane blebbing, in the absence of caspase activity in TNF-treatedmurine WEHI-S fibrosarcoma cells. Furthermore, the depletion of heatshock protein 70 (Hsp70) in various human cancer cells as well assupraoptimal activation of T cells triggers LMP and cathepsin-mediatedapoptosis-like PCD without the activation of the intrinsic apoptosispathway. In line with these data, cathepsin B can induce nuclearapoptosis in isolated nuclei. Thus, cathepsins appear to carry both theability to act as initiator—as well as effector proteases of programmedcell death depending on the stimulus and the cellular context.Especially their ability to mediate PCD in cancer cells, where themitochondrial death pathway is blocked for example due to overexpressionof Bcl-2, raises hopes that treatments inducing LMP may prove effectivein treatment of cancers that are resistant to inducers of classisapoptosis. This idea is further supported by data showing thatimmortalization and transformation can sensitize cells to the lysosomalcell death.

Signaling to LMP

As described above, LMP followed by the release of lysosomal contents,especially cathepsins, to the cytosol is considered to be the keyactivation step of the lysosomal death pathway. However, the signalingpathways leading to LMP are still only beginning to emerge. One of thebest studied mechanisms is the signaling from the tumor necrosis factorreceptor 1 although the clarification of this signaling pathway to LMPhas been greatly complicated by widely different responses in differenttarget cells.

In summary, TNF can either induce caspase-dependent or -independent LMPdepending on cellular context. In addition, the TNF-related ligandsFasL, TRAIL and TWEAK have also all been associated withcaspase-independent PCD with either apoptotic or necrotic morphology.Pharmacological and genetic studies indicate that the caspase-mediatedpathway leading from TNF to LMP is dependent on caspases-8 and -9,although activation of caspase-9 differs widely between human and murinecells. The link between caspases and LMP is as yet unknown, and althoughTNF-induced caspase-8-mediated cleavage of Bid has been suggested tocontribute to LMP, these findings could not be verified by TNF-inducedLMP in Bid-deficient iMEFs. Bid has furthermore been suggested to be atarget for cathepsins in lysosomal death pathways implicating Biddownstream, rather than upstream, of the LMP.

TNF also stimulates sphingomyelin breakdown to phosphorylcholine andceramide by activating neutral sphingomyelinase (SMase) at the plasmamembrane and acid or acidic SMase (aSMase) in the lysosomal compartment.Both events have been implicated in TNF-induced cell death pathways, butso far only neutral SMase has been connected to LMP through the factorassociated with neutral SMase (FAN). Studies based on FAN deficientiMEFs as well as human fibroblasts expressing a dominant negative formof FAN have shown that FAN does not only mediate TNF-induced ceramideproduction, but also contributes to the caspase-8 processing and celldeath. Since the TNF-induced LMP in murine hepatocytes depends oncaspase-8, its reduced processing may explain the reduced LMP inTNF-treated hepatocytes expressing dominant negative FAN. The role ofceramide and its metabolites can, however, not be ruled out. Their rolein TNF-induced death signaling is supported by the reduced TNF andFas-induced hepatotoxicity in mice deficient for aSMase, which isactivated downstream of caspase-8. Especially sphingosine that isgenerated from ceramide in a reaction catalyzed by the lysosomal enzymeacid ceramidase is a tempting candidate, as it, contrary to ceramide,can act as a detergent, directly destabilizing the lysosomal membrane.In addition to increasing the generation of the sphingosine precursor,ceramide, by activating SMases, TNF regulates sphingosine levels also bycathepsin B-mediated downregulation of sphingosine kinase-1, en enzymethat converts the pro-apoptotic sphingosine to an anti-apoptoticsphingosine-1-phospate. This activity of cathepsin B could result in theaccumulation of sphingosine in the lysosomes and may thus, at leastpartially, explain the requirement of cathepsin B for an efficient LMPin TNF-treated hepatocytes.

TNF can also trigger LMP and cell death in the presence of caspaseinhibitors. This pathway is independent of caspase-8, but requires thedeath domain-containing receptor interacting protein-1 (RIP-1) andinvolves the generation of reactive oxygen species. Oxidative stresscan, together with intra-lysosomal iron, generate oxygen radicalsthrough a Fenton-type chemistry and thereby may cause oxidation oflysosomal membrane lipids, resulting in the destabilization of themembrane and the release of the lysosomal content. The molecular linksbetween RIP-1. oxidative stress and LMP are, however, still missing.

The induction of cell death by several classic apoptosis inducers (e.g.p53, etoposide and staurosporine) also involves LMP followed bycathepsin-dependent mitochondrial membrane permeabilization. However,the signaling pathways from these stimuli to LMP remain to be revealed.

Cellular Defense Mechanisms Against LMP

Given the potential fatal outcome of LMP, it is not surprising thatcells have developed numerous strategies to counteract it—either byinhibiting the LMP itself or by protecting cells against the acidhydrolases leaking to the cytosol as a consequence of LMP.

Among its many other functions, phosphatidylinositol 3-kinase (PI3K) hasbeen reported to protect lysosomes against destabilization. Inhibitionof PI3K in human vascular endothelial cells induces the release ofcathepsin B to the cytosol arguing for a rather direct role of PI3K inpreserving lysosomal membrane integrity. Furthermore, PI3K inhibitorssensitize the cells to the TNF- and interleukin-1-induced lysosomaldeath pathways. Altered lysosomal functions and increased expressionlevels of cathepsins in cancer cells may pose a threat in form ofdecreased stability of lysosomes. Thus, PI3K, which is commonlyactivated in human cancer cells, may also contribute to lysosomalstability of tumor cells and thereby increase their cell deathresistance. Whereas the role of PI3K on the stability of tumor celllysosomes is purely speculative, recent data advocate for a role forHsp70 in the protection of lysosomes against membrane-disruptivestimuli. This work has been mainly done in tumor cells, which also oftendemonstrate a localization of Hsp70 on the plasma membrane as well as inthe endolysosomal compartment.

In the event of release of lysosomal proteases to the cytosol upon LMP,cytosolic protease inhibitors present a bulwark against its deleteriousconsequences. Whereas no endogenous inhibitors of cathepsin D are known,cysteine cathepsins can be effectively inhibited by at least threecytosolic protease inhibitors, i.e. cystatin A and B and serine proteaseinhibitor 2A (Spi2A) which was recently found to possess potentinhibitor activity also against several cysteine cathepsins (B, H, K, Land V) and cathepsin G. The importance of these inhibitors in preventingPCD in physiological and pathological conditions is demonstrated bycystatin B-deficient mice which display increased apoptosis ofcerebellar granule cells. Moreover, the expression of Spi2A is inducedupon TNF-treatment via the NF-κB pathway, and effectively inhibitsTNF-induced cytosolic cathepsin B activity and cell death in MEFs.Interestingly, it has just been reported that in C. Elegans, thecytosolic serine protease inhibitor (serpin)-6 can protect against boththe induction as well as the lethal effects from lysosomal injury causedby hypo-osmotic stress as well as a variety of other lysosomal stresses,demonstrating that protection against LMP is an evolutionarily conservedmechanism.

Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) are a group of approximately 40 rareinherited metabolic disorders that result from defects in lysosomalfunction. LSDs are caused by lysosomal dysfunction usually as aconsequence of deficiency of a single enzyme required for the metabolismof lipids, glycoproteins or mucopolysaccharides. Although each disorderresults from different gene mutations that translate into a deficiencyin enzyme activity, they all share a common biochemicalcharacteristic—all lysosomal disorders originate from an abnormalaccumulation of substances inside the lysosome.

Individually, LSDs occur with incidences of less than 1:100.000,however, as a group the incidence is about 1:5000-1:10.000. Most ofthese disorders are autosomal recessively inherited, however a few areX-linked recessively inherited, such as Fabry disease.

The lysosomal storage diseases are generally classified by the nature ofthe primary stored material involved, and can be broadly broken into thefollowing:

-   -   lipid storage disorders (or lipidoses), mainly sphingolipidoses        (including Gaucher's and Niemann-Pick diseases)        -   gangliosidosis (including Tay-Sachs disease)        -   leukodystrophies    -   mucopolysaccharidoses (including Hunter syndrome and Hurler        disease)    -   glycoprotein storage disorders (glycoproteinosis)    -   mucolipidoses

Depending on the severity of the disease patients either die at a youngand unpredictable age, many within a few months or years of birth,whereas others survive into early adulthood finally succumbing to thevarious pathologies of their particular disorder. The symptoms of LSDvary, depending on the particular disorder and can be mild to severe.They can include developmental delay, movement disorders, seizures,dementia, deafness and/or blindness. Some people with LSD have enlargedlivers (hepatomegaly) and enlarged spleens (splenomegaly), pulmonary andcardiac problems, and abnormal bone growth.

The majority of patients are initially screened by an enzyme assay,which is the most efficient method to arrive at a definitive diagnosis.In some families where the disease-causing mutation(s) is known and incertain genetic isolates, mutation analysis may be performed. As theremay be numerous different mutations, sequencing of the gene encoding theparticular affected enzyme is sometimes necessary to confirm thediagnosis. Prenatal diagnosis may be useful when there is a knowngenetic risk factor.

The present invention is in one embodiment related to a method fortreating lysosomal storage disorders.

Lysosomal Sphinolipid Hydrolysis

A multitude of enzymes are involved in the lysosomal catabolism ofsphingolipids (or glycophingolipids) (see FIG. 4). These enzymes, ormore specifically hydrolases, are each responsible for the degradationof a specific sphingolipid.

The lysosomal sphingolipid hydrolases interacts with sphingolipidactivator proteins (SAP or saposins) to stimulate the activity of saidhydrolases. SAPs are considered to facilitate the enzyme/substrateinteraction between water-soluble enzymes and membrane-bound substrates.

Further, the lipid composition of late endosomal and lysosomalcompartments are characterized by the presence of negatively chargedphospholipids such as BMP and PI (phosphatidylinositol), which alsostimulates the activity of some hydrolases. The BMP-dependent lysosomalhydrolases include sialidase, α-galactosidase A, glucosylceramidase,β-galactosylceramidase, arylsulfatase A, acid ceramidase andSphingomyelinase.

Co-Factor Saposins

Saposins are small lysosomal proteins that serve as activators ofvarious lysosomal lipid-degrading enzymes. They probably act byisolating the lipid substrate from the membrane surroundings, thusmaking it more accessible to the soluble degradative enzymes. Allmammalian saposins are synthesized as a single precursor molecule(prosaposin) which contains four Saposin-B domains, yielding the activesaposins after proteolytic cleavage, and two Saposin-A domains that areremoved in the activation reaction. The Saposin-B domains also occur inother proteins, many of them active in the lysis of membranes.

Prosaposin (PSAP) is a protein which in humans is encoded by the PSAPgene. This gene encodes a highly conserved glycoprotein which is aprecursor for 4 cleavage products: saposin A, B, C, and D. Saposin is anacronym for Sphingolipid Activator Protein or SAP. Each domain of theprecursor protein is approximately 80 amino acid residues long withnearly identical placement of cysteine residues and glycosylation sites.Saposins A-D localize primarily to the lysosomal compartment where theyfacilitate the catabolism of glycosphingolipids with shortoligosaccharide groups. The precursor protein exists both as a secretoryprotein and as an integral membrane protein and has neurotrophicactivities. Saposins A-D are required for the hydrolysis of certainshingolipids by specific lysosomal hydrolases.

The saposins are important co-activators of sialidase (SAP-B),α-galactosidase A (SAP-B), glucosylceramidase (SAP-C),3-galactosylceramidase (SAP-C), arylsulfatase A (SAP-B) and acidceramidase (SAP-D). Acidic sphingomyelinase (aSMase) is not criticallydependent on any of the known activator proteins, however the presenceof saposins increases the activity of this enzyme. A fifth saposin;GM2-activator protein has also been characterised.

BMP

Bis(monoacylglycero)phosphate (BMP), also known as Lysobisphosphatidicacid, is a major part of the lipid composition of late endosomal andlysosomal compartments. It is a negatively charged phospholipid, morespecifically a glycerol-phospholipid.

BMP was first isolated from rabbit lung but is now known to be a commonif minor constituent of all animal tissues. Its stereochemicalconfiguration differs from that of other animal glycero-phospholipids inthat the phosphodiester moiety is linked to positions sn-1 and sn-1′ ofglycerol, rather than to position sn-3. It remains unclear whetherpositions sn-3 and 3′ or sn-2 and sn-2′ in the glycerol moieties areesterified with fatty acids. Whatever the positions of the fatty acidson the glycerol molecule, their compositions can be distinctive with18:1(n−9) and 18:2(n−6), 20:4 and 22:6(n−3) being abundant, althoughthis is highly dependent on the specific tissue, cell type or organelle.Such distinctive compositions suggest quite specific functions, some ofwhich have yet to be revealed.

BMP is usually a rather minor component of animal tissues. However, itis highly enriched in the lysosomes of liver and other tissues, where itcan amount to 15% or more of the membrane phospholipids, and it is nowrecognized as a marker for this organelle. It is the late endosomes andthe lysosomes that contain the unique lipid, BMP. Indeed, there appearto be internal membranes of the late endosomes that contain as much as70% of the phospholipids as BMP.

If the reported presence of BMP in some alkalophilic strains of Bacillusspecies can be confirmed, this will be the only known exception to therule that this lipid is strictly of mammalian origin and not present inprokaryotes, yeasts and higher plants.

There is good evidence that BMP is synthesised fromphosphatidylglycerol, primarily in the endosomal system. In what isbelieved to be the primary route, a phospholipase A2 removes the fattyacid from position sn-2 of phosphatidylglycerol in the first step. Inthe second step, the lysophosphatidylglycerol is acylated on the sn-2′position of the head group glycerol moiety to yield sn-3:sn-1′lysobisphosphatidic acid, by means of a transacylase reaction withlysophosphatidylglycerol as both the acyl donor and acyl acceptor. Thethird step has still to be adequately described but must involve removalof the fatty acid from position sn-1 of the primary glycerol unit and arearrangement of the phosphoryl ester from the sn-3 to the sn-1position. Finally position sn-2 of the primary glycerol unit isesterified, probably by a transacylation reaction with anotherphospholipid as donor (hence the distinctive fatty acid compositions).Other biosynthetic routes may be possible.

The function of BMP in lysosomes is under active investigation. It mayhave a structural role in developing the complex intraluminal membranesystem, aided by a tendency not to form a bilayer. It is a cone-shapedmolecule, and it encourages fusion of membranes at the pH in theendosomes. Further, its unique stereochemistry means that it isresistant to phospholipases, so it will hinder or prevent self digestionof the lysosomal membranes. The fatty acid constituents may turn overrapidly by transacylation, but the glycerophosphate backbone is stable.A further possibility is that this lipid may associate with specificproteins in membrane domains, functionally similar to rafts. It has beensuggested that that the characteristic network of BMP-rich membranescontained within multivesicular late endosomes regulates cholesteroltransport by acting as a collection and re-distribution point. Forexample, when lysosomal membranes are incubated with antibodies to BMP,cholesterol tends to accumulate. The process is under the control ofAlix/AlP1, which is a protein that interacts specifically with BMP andis involved in sorting into multivesicular endosomes.

BMP is known to greatly stimulate the enzymes involved in thedegradation of glycosylceramides, such as the sphingolipid activatorproteins like the saposins. In this instance, it may simply function toprovide a suitable environment for the interaction of theglycosphingolipid hydrolases and their activator. In addition, it has adynamic role in the provision of arachidonate for eicosanoid productionin alveolar macrophages.

For BMP-dependent enzymes, the rate of hydrolysis is increaseddramatically when BMP is present in the membrane, for aSMase evenwithout the presence of an activator protein such as saposin. In FIG. 4,a stippled circle marks the enzymes, or the disease in which this enzymeis defect, which show a dependence on BMP.

BMP is involved in the pathology of lysosomal storage diseases such asNiemann-Pick C disease (cholesterol accumulation) and certaindrug-induced lipidoses. In these circumstances, its composition tends tochange to favour molecular species that contain less of thepolyunsaturated components. It is an antigen recognized by autoimmunesera from patients with a rare and poorly understood disease known asantiphospholipid syndrome, so it is probably a factor in thepathological basis of this illness.

The present invention is in one embodiment related to a method fortreating lysosomal storage disorders, by exploiting the interactionbetween Hsp70 and BMP.

The Lipid Storage Disorders

Lipid storage disorders (or lipidoses) are a subgroup of the lysosomalstorage disorders in which harmful amounts of lipids accumulate in theintracellular space due to reduced expression or function of the enzymesneeded to metabolize lipids. Over time, this excessive storage of lipidscan cause permanent cellular and tissue damage, particularly in thebrain. peripheral nervous system, liver, spleen and bone marrow.

Lipids are a broad group of naturally-occurring molecules which includesfats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E andK), monoglycerides, diglycerides, phospholipids, and others. The mainbiological functions of lipids include energy storage, as structuralcomponents of cell membranes, and as important signaling molecules.

Lipids may be broadly defined as hydrophobic or amphiphilic smallmolecules; the amphiphilic nature of some lipids allows them to formstructures such as vesicles, liposomes, or membranes in an aqueousenvironment. Biological lipids originate entirely or in part from twodistinct types of biochemical subunits: ketoacyl and isoprene groups.Using this approach, lipids may be divided into eight categories: fattyacyls, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids and polyketides (derived from condensation of ketoacylsubunits); and sterol lipids and prenol lipids (derived fromcondensation of isoprene subunits).

Although the term lipid is sometimes used as a synonym for fats, fatsare a subgroup of lipids called triglycerides. Lipids also encompassmolecules such as fatty acids and their derivatives (including tri-,di-, and monoglycerides and phospholipids), as well as othersterol-containing metabolites such as cholesterol.

Several lysosomal storage disorders characterized by the accumulation oflipids (i.e., lipid storage disorders) have been characterized; theseare outlined herein below.

The present invention is in one embodiment related to a method fortreating lipid storage disorders.

Niemann-Pick Disease

Niemann-Pick disease (NPD) is caused by a defect in the acidicsphingomyelinase enzyme (aSMase), with the systematic name sphingomyelinphosphodiesterase. The bulk of membrane sphingomyelin is hydrolysed bythe lysosomal enzyme aSMase to produce ceramide (and phosphocholine).Sphingomyelin consists of a ceramide membrane anchor which is linked toa short hydrophilic phosphorylcholine moiety.

Sphingomyelinase is not critically dependent on any of the knownactivator proteins, making the assumed intramolecular activator domainof aSMase and the presence of negatively charged lipids in the lysosomessufficient for sphingomyelin turnover, aSMase thus does not require thepresence of saposins as a co-factor; however the presence of saposinsinvariably further stimulates the activity of this enzyme. (Ferlinz etal., 1999). aSMase activity is stimulated by BMP.

When sphingomyelin cannot be metabolized properly it is accumulatedwithin the cell, eventually causing cell death and the malfunction ofmajor organ systems. Symptoms may include lack of muscle coordination,brain degeneration, learning problems, loss of muscle tone, increasedsensitivity to touch, spasticity, feeding and swallowing difficulties,slurred speech, and an enlarged liver and spleen. There may be cloudingof the cornea and a characteristic cherry-red halo develops around thecenter of the retina.

Niemann-Pick disease (NPD) has 4 related types; types A, B, C and D. Alltypes of NPD are inherited in an autosomal recessive pattern and canaffect both males and females. In types A and B, insufficient activityof the enzyme aSMase causes the build up of toxic amounts ofsphingomyelin. The disease occurs when both copies of a person's aSMasegene (both alleles) have a mutation.

Niemann-Pick Type A (NPDA), the most common type, occurs in infants. Itis characterized by jaundice, an enlarged liver, and profound braindamage. There is currently no effective treatment for persons with typeA, and patients with type A die in infancy, usually before the age of 18months.

Niemann-Pick Type B (NPDB) involves an enlarged liver and spleen, whichusually occurs in the pre-teen years, and respiratory problems arecommon. The enlargement of organs and the respiratory problems can causecardiovascular stress and can lead to heart disease later in life.Patients with NPDB generally have little or no neurologic involvement.Bone marrow transplantation has been attempted in a few patients withtype B, and mixed results have been reported. The future development ofenzyme replacement and gene therapies might also be helpful for thosewith type B. Children with Type B may live a comparatively long time,but may require supplemental oxygen because of lung impairment.

NPDA and NPDB are both caused by the same enzymatic deficiency and thereis growing evidence that the two forms represent opposite ends of acontinuum. People with NPDA generally have little or no aSMaseproduction (less than 1% of normal) while those with NPDB haveapproximately 10% of the normal level of aSMase.

There are approximately 1,200 cases of NPA and NPB world wide with themajority being Type B or an intermediate form.

NPDA and NPDB are diagnosed by measuring the level of activity of aSMasein white blood cells from a blood sample. While this test will identifypersons with Type and B, it is not very reliable for detecting personswho are carriers (who have only one non-functional copy of the ASMgene). Further, the test will show decreased activity of aSMase, but itcannot always predict whether the individual will have type A or Type Bor an intermediate variant of the disease; that requires clinicalevaluation of the individual.

In certain populations, specific mutations account for a high percentageof cases of aSMase deficiency. For NPDA, the mutations R496L, fsP330 andL302P account for over 95% of disease-causing genetic changes in theAshkenazi Jewish population. Direct testing of individuals in thispopulation for these 3 changes is used for carrier identification. Inother populations, the mutations must first be identified in theaffected individual before DNA carrier testing can be performed forfamily members.

For NPDB, the H421Y and K576N aSMase mutations account for 85% of theSaudi Arabian NPDB population; the L137P, fsP189 and L549P mutationsaccount for 75% of the Turkish NPDB population; the S379P, R441X andR474W mutations account for 55% of the Portuguese NPDB population; theA196P mutations account for 42% of the English/Scottish NPDB population,and the mutations F480L and DeltaR608 have also been identified asdisease-causing in NPDB patients.

Niemann-Pick Type C (NPDC) is very different than Type A or B. NPDCPatients are not able to metabolize cholesterol and other lipidsproperly within the cell, and is characterized by a defect that disruptsthe transport of cholesterol between brain cells. Consequently,excessive amounts of cholesterol and other lipids accumulate within theliver, spleen and brain. NPDC causes a secondary reduction of aSMaseactivity, which led all three types to be considered forms of the samedisease.

There is considerable variation in when Type C symptoms first appear andin the progression of the disease. Symptoms may appear as early as a fewmonths of age or as late as adulthood. Vertical gaze palsy (theinability to move the eyes up and down), enlarged liver, enlargedspleen, or jaundice in young children are strong indications that NPCshould be considered. It is common for only one or two symptoms toappear in the early stages of the disease. In most cases, neurologicalsymptoms begin appearing between the ages of 4 and 10. Generally, thelater neurological symptoms begin, the slower the progression of thedisease.

Type C Niemann-Pick disease has about 500 cases diagnosed worldwide. Itis believed, however, that the number of people affected by NPDC ishigher, but diagnostic difficulties do not allow an accurate assessmentof the occurrence rate. NPDC has been initially diagnosed as a learningdisability, mild retardation, clumsiness, and delayed development offine motor skills.

Niemann-Pick Type D is now considered a variant of type C. Type Dusually occurs in people with an ancestral background in Nova Scotia.Individuals with types C and D are frequently placed on alow-cholesterol diet, but its clinical benefit is not convincing. Thelife expectancy of persons with types C and D varies, however thedisease is always fatal. The vast majority of children die before age20.

NPDC is a rare and extremely variable condition and therefore may not berecognized by some health care providers. For those specialists who dosuspect this diagnosis in a patient, it can be determined by taking askin biopsy, culturing the fibroblasts, and studying their ability totransport and store cholesterol. The transport of cholesterol in thecells is studied by measuring conversion of the cholesterol from oneform to another (esterification). The storage of cholesterol is assessedby staining the cells with a chemical (filipin) that glows underultraviolet light.

In 1997, the NPC1 gene was identified. Mutations, or disease-causingchanges, in this gene are responsible for about 95% of all NPDC cases.Since then, over 250 different genetic mutations related to NPDC havebeen identified in this gene and in the second NPDC gene, called NPC2.Overall, in about 95% of cases, it is possible to identify the geneticchanges that have caused the disease if the diagnosis of NPC has firstbeen confirmed by the testing outlined above. However, because there areso many unique mutations in these genes, and there are patients withclassic NPC in whom mutations have not been identified, it is notoptimal to use genetic testing as a general diagnostic tool for NPDC.

Niemann-Pick Disease affects all segments of the population with casesreported from North America, South America, Europe. Africa, Asia, andAustralia. However a higher incidence has been found in certainpopulations:

-   -   Ashkenazi Jewish population (NPDA and NPDB)    -   French Canadian population of Nova Scotia (type D—now considered        a variant of NPDC)    -   Maghreb region (Tunisia, Morocco, and Algeria) of North Africa        (NPDB)    -   Spanish-American population of southern New Mexico and Colorado        (NPDC)

The present invention is in one embodiment related to a method fortreating Niemann-Pick disease, by modulation of acidic sphingomyelinaseenzyme (aSMase) activity.

Farber Disease

Farber disease is caused by a defect in the acid ceramidase enzyme. Acidceramidase is responsible for the conversion of ceramide to sphingosine(and fatty acid); the defect thus leads to an accumulation of ceramide.Its activity is stimulated by BMP and is dependent on saposins.

Acid ceramidase is also known as N-acylsphingosine amidohydrolase, andis coded by the gene ASAH1. It is a heterodimeric protein consisting ofa nonglycosylated alpha subunit and a glycosylated beta subunit that iscleaved to the mature enzyme posttranslationally.

Farber disease is also known as Farber's lipogranulomatosis, ceramidasedeficiency, Fibrocytic dysmucopolysaccharidosis, and Lipogranulomatosis.It is an extremely rare autosomal recessive disease characterized byabnormalities in the joints, liver, throat, tissues and central nervoussystem. The liver, heart, and kidneys may also be affected. Symptoms aretypically seen in the first few weeks of life and include impaired motorand mental ability and difficulty with swallowing. Other symptoms mayinclude arthritis, swollen lymph nodes and joints, hoarseness, nodulesunder the skin (and sometimes in the lungs and other parts of the body),chronic shortening of muscles or tendons around joints, and vomiting.Affected persons may require the insertion of a breathing tube. Insevere cases, the liver and spleen are enlarged.

Currently there is no specific treatment for Farber disease.Corticosteroids can help relieve pain. Nodes can be treated with bonemarrow transplants, in certain instances, or may be surgically reducedor removed. Most children with the classic form of Farber's disease dieby age 2, usually from lung disease. Individuals having a milder form ofthe disease may live into their teenage years.

The present invention is in one embodiment related to a method fortreating Farber disease, by modulation of acid ceramidase enzymeactivity.

Krabbe Disease

Krabbe disease is caused by a defect in the β-galactosylceramidaseenzyme. β-galactosylceramidase is responsible for the conversion ofgalactosylceramide to ceramide; the defect thus leads to an accumulationof galactosylceramide. Its activity is stimulated by BMP and isdependent on saposins.

Krabbe disease is also known as globoid cell leukodystrophy orgalactosylceramide lipidosis. It is a rare, often fatal degenerativeautosomal recessive disorder that affects the myelin sheath of thenervous system. It occurs in about 1 in 100,000 births. A higherprevalence, about 1 in 6,000 has been reported in some Arab communitiesin Israel.

Krabbe disease is caused by mutations in the GALC gene, which causes adeficiency of the galactosylceramidase enzyme. The lipid buildup affectsthe growth of the nerve's protective myelin sheath (the covering thatinsulates many nerves) and causes severe degeneration of motor skills.

Infants with Krabbe disease are normal at birth. Symptoms begin betweenthe ages of 3 and 6 months with irritability, fevers, limb stiffness,seizures, feeding difficulties, vomiting, and slowing of mental andmotor development. In the first stages of the disease, doctors oftenmistake the symptoms for those of cerebral palsy. Other symptoms includemuscle weakness, spasticity, deafness, optic atrophy and blindness,paralysis, and difficulty when swallowing. Prolonged weight loss mayalso occur. There are also juvenile- and adult-onset cases of Krabbedisease, which have similar symptoms but slower progression. In infants,the disease is generally fatal before age 2. Patients with late-onsetKrabbe disease tend to have a slower progression of the disease and livesignificantly longer.

Although there is no cure for Krabbe disease, bone marrowtransplantation has been shown to benefit cases early in the course ofthe disease. Generally, treatment for the disorder is symptomatic andsupportive. Physical therapy may help maintain or increase muscle toneand circulation. A recent study reports that cord blood transplants havebeen successful in stopping the disease as long as they are given beforeovert symptoms appear.

The present invention is in one embodiment related to a method fortreating Krabbe disease, by modulation of β-galactosylceramidase enzymeactivity.

Fabry Disease

Fabry disease is caused by a defect in the α-galactosidase A enzyme.α-galactosidase A is responsible for the conversion ofglobotriaosylceramide to lactosylceramide; the defect thus leads to anaccumulation of globotriaosylceramide (also abbreviated as Gb3, GL-3, orceramide trihexoside). Its activity is stimulated by BMP and isdependent on saposins.

Fabry disease is also known as Anderson-Fabry disease, Angiokeratomacorporis diffusum, Ruiter-Pompen-Wyers syndrome, Ceramidetrihexosidosis, and Sweeley-Klionsky disease. It is an X-linkedrecessive (inherited) disease that affects hemizygous males, as well asboth heterozygous and homozygous females; males tend to experience themost severe clinical symptoms, while females vary from virtually nosymptoms to those as serious as males. This variability is thought to bedue to X-inactivation patterns during embryonic development of thefemale.

Symptoms include anhidrosis (lack of sweating), fatigue, angiokeratomas(benign cutaneous injury of capillaries), burning extremity pain andocular involvement. Angiokeratomas are tiny, painless papules thatappear at any region of the body, but are predominant on the thighs,buttocks, lower abdomen, and groin. Cosmetic ocular involvement may bepresent showing cornea verticillata (also known as vortex keratopathy).Keratopathy may be the presenting feature in asymptomatic carriers, andmust be differentiated from other causes of vortex keratopathy (e.g.drug deposition in the cornea). Other ocular findings that can be seeninclude conjunctival aneurysms, posterior spoke-like cataracts,papilloedema, macular edema, optic atrophy and retinal vasculardilation. Kidney complications are a common and serious effect of thedisease; renal insufficiency and renal failure may worsen throughoutlife. Proteinuria is often the first sign of kidney involvement. Cardiaccomplications may also occur; heart related effects worsen with age andmay lead to increased risk of heart disease. Cerebrovascular effectslead to an increased risk of stroke. Other symptoms include tinnitus,vertigo, nausea, and diarrhea.

Symptoms are typically first experienced in early childhood and can bevery difficult to understand; the rarity of Fabry disease to manyclinicians sometimes leads to misdiagnoses or ignorance. Manifestationsof the disease usually increase in number and severity as an individualage.

Until recently, treatment of Fabry disease targeted the symptomaticeffects. However, it is currently being treated at the cellular levelthrough enzyme replacement therapy (ERT) using Agalsidase alpha(Replagal) and Agalsidase beta (Fabrazyme). The cost of these drugs isproblematic (approximately $250,000 US a year/patient) and remains abarrier to many patients in some countries. Enzyme replacement therapy(typically infused every two weeks) may be performed in the patient'shome by the patients themselves. Enzyme replacement therapy is not acure, and it must be infused recurrently for maximum benefit.

The present invention is in one embodiment related to a method fortreating Fabry disease, by modulation of α-galactosidase A enzymeactivity.

Gaucher Disease

Gaucher disease is caused by a defect in the glucosylceramidase enzyme(also known as glucocerebrosidase and acid 3-glucosidase); a 55.6 KD,497 amino acids long protein. Glucosylceramidase is responsible for theconversion of glycosylceramide (or glucocerebroside) to ceramide; thedefect thus leads to an accumulation of glycosylceramide. Its activityis stimulated by BMP and is dependent on saposins.

Gaucher's disease is the most common of the lysosomal storage diseases.Fatty material can collect in the spleen, liver, kidneys, lungs, brainand bone marrow. Symptoms may include enlarged spleen and liver, livermalfunction, skeletal disorders and bone lesions that may be painful,severe neurologic complications, swelling of lymph nodes and(occasionally) adjacent joints, distended abdomen, a brownish tint tothe skin, anemia, low blood platelets and yellow fatty deposits on thesclera. Persons affected most seriously may also be more susceptible toinfection.

The disease shows autosomal recessive inheritance and therefore affectsboth males and females. Different mutations of glucosylceramidasedetermine the remaining activity of the enzyme, and, to a large extent,the phenotype. Research suggests that heterozygotes for particularglucosylceramidase mutations are at an increased risk of Parkinson'sdisease and particular malignancies (non-Hodgkin lymphoma, melanoma andpancreatic cancer).

Glycosylceramide is a cell membrane constituent of red and white bloodcells. The macrophages that clear these cells are unable to eliminatethe waste product, which accumulates in fibrils, and turn into Gauchercells, which appear on light microscopy to resemble crumpled-up paper.

Gaucher's disease has three common clinical subtypes. Each type has beenlinked to particular mutations. In all, there are about 80 knownmutations.

-   -   Type I (or nonneuropathic type) is the most common form of the        disease, occurring in approximately 1 in 50,000 live births. It        occurs most often among persons of Ashkenazi Jewish heritage,        100 times the occurrence in the general populace. Symptoms may        begin early in life or in adulthood and include enlarged liver        and grossly enlarged spleen (together hepatosplenomegaly); the        spleen can rupture and cause additional complications. Skeletal        weakness and bone disease may be extensive. Spleen enlargement        and bone marrow replacement cause anemia, thrombocytopenia and        leukopenia. The brain is not affected, but there may be lung        and, rarely, kidney impairment. Patients in this group usually        bruise easily (due to low levels of platelets) and experience        fatigue due to low numbers of red blood cells. Depending on        disease onset and severity, type 1 patients may live well into        adulthood. Many patients have a mild form of the disease or may        not show any symptoms.    -   Type II (or acute infantile neuropathic Gaucher's disease)        typically begins within 6 months of birth and has an incidence        rate of approximately 1 in 100,000 live births. Symptoms include        an enlarged liver and spleen, extensive and progressive brain        damage, eye movement disorders, spasticity, seizures, limb        rigidity, and a poor ability to suck and swallow. Affected        children usually die by age 2.    -   Type III (the chronic neuropathic form) can begin at any time in        childhood or even in adulthood, and occurs in approximately 1 in        100,000 live births. It is characterized by slowly progressive        but milder neurologic symptoms compared to the acute or type 2        version. Major symptoms include an enlarged spleen and/or liver,        seizures, poor coordination, skeletal irregularities, eye        movement disorders, blood disorders including anemia and        respiratory problems. Patients often live into their early teen        years and adulthood.

The National Gaucher Foundation states that around 1 in 100 people inthe general U.S. population is a carrier for type 1 Gaucher's disease,giving a prevalence of 1 in 40,000; among Ashkenazi Jews the rate ofcarriers is considerably higher, at roughly 1 in 15. Type 2 Gaucher'sdisease shows no particular preference for any ethnic group. Type 3Gaucher's disease is especially common in the population of the NorthernSwedish region of Norrbotten where the incidence of the disease is 1 in50,000.

For type 1 and most type 3 patients, enzyme replacement treatment withintravenous recombinant glucosylceramidase can decrease liver and spleensize, reduce skeletal abnormalities, and reverse other manifestations.The rarity of the disease means that dose-finding studies have beendifficult to conduct, so there remains controversy over the optimal doseand dosing frequency. Due to the low incidence, this has become anorphan drug in many countries. The currently existing treatment ofGaucher's disease, Cerezyme® (imiglucerase for injection), costs up to$550,000 annually for a single patient and the treatment should becontinued for life. Miglustat is another drug approved for this diseasein 2003.

Successful bone marrow transplantation cures the non-neurologicalmanifestations of the disease, because it introduces a monocytepopulation with active glucosylceramidase. However, this procedurecarries significant risk and is rarely performed in Gaucher patients.Surgery to remove the spleen (splenectomy) may be required on rareoccasions if the patient is anemic or when the enlarged organ affectsthe patient's comfort. Blood transfusion may benefit some anemicpatients. Other patients may require joint replacement surgery toimprove mobility and quality of life. Other treatment options includeantibiotics for infections, antiepileptics for seizures, bisphosphonatesfor bone lesions, and liver transplants.

Substrate reduction therapy may prove to be effective in stopping Type2, as it can cross through the blood barrier into the brain. There iscurrently no effective treatment for the severe brain damage that mayoccur in patients with types 2 and 3 Gaucher disease.

The present invention is in one embodiment related to a method fortreating Gaucher disease, by modulation of glucosylceramidase enzymeactivity.

Sialidosis

Sialidosis, or Mucolipidosis type I (ML I), is caused by a defect in thesialidase enzyme (or alpha-neuraminidase). Sialidase is responsible forthe conversion of GM3 to lactosylceramide; the defect thus leads to anaccumulation of GM3. Its activity is stimulated by BMP and is dependenton saposins.

Sialidosis is inherited in an autosomal recessive manner. Symptoms areeither present at birth or develop within the first year of life. Inmany affected infants, excessive swelling throughout the body is notedat birth. These infants are often born with coarse facial features, suchas a flat nasal bridge, puffy eyelids, enlargement of the gums, andexcessive tongue size (macroglossia). Many infants with are also bornwith skeletal malformations such as hip dislocation. Infants oftendevelop sudden involuntary muscle contractions (called myoclonus) andhave red spots in their eyes (cherry red macules). They are often unableto coordinate voluntary movement (called ataxia). Tremors, impairedvision, and seizures also occur. Tests reveal abnormal enlargement ofthe liver (heptomegaly) and spleen (splenomegaly) and extreme abdominalswelling. Infants generally lack muscle tone (hypotonia) and have mentalretardation that is either initially or progressively severe. Manypatients suffer from failure to thrive and from recurrent respiratoryinfections. Most infants with ML I die before the age of 1 year.

Sialidosis may be sub-categorised according to the age at which symptomsbegin and the types of symptoms present. The effects of the disease mayrange from mild to severe.

Sialidosis is a rare disorder that has no racial predilection. Verylittle population data are available, but a study from the Netherlandsreported a frequency of approximately 1 case in 2,175,000 live births.However, this rate may not apply to all populations, some of which couldhave a higher incidence; moreover, missed clinical recognition is animportant factor when newborn screening is not an option.

Treatment options for sialidosis remain limited and are primarilydirected at supportive care and symptomatic relief.

The present invention is in one embodiment related to a method fortreating Sialidosis, by modulation of sialidase activity.

Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) or Arylsulfatase A deficiency iscaused by a defect in the arylsulfatase A enzyme (orcerebroside-sulfatase). Arylsulfatase A is responsible for theconversion of sulfatide (or cerebroside 3-sulfate) togalactosylceramide; the defect thus leads to an accumulation ofsulfatide. Its activity is stimulated by BMP and is dependent onsaposins.

It is a lysosomal storage disease which is commonly listed in the familyof leukodystrophies. Leukodystrophiea affect the growth and/ordevelopment of myelin, the fatty covering which acts as an insulatoraround nerve fibers throughout the central and peripherial nervoussystems.

Like many other genetic disorders that affect lipid metabolism, thereare several forms of MLD, which are late infantile, juvenile, and adult:

-   -   In the late infantile form, which is the most common form MLD,        affected children begin having difficulty walking after the        first year of life. Symptoms include muscle wasting and        weakness, muscle rigidity, developmental delays, progressive        loss of vision leading to blindness, convulsions, impaired        swallowing, paralysis, and dementia. Children may become        comatose. Untreated, most children with this form of MLD die by        age 5, often much sooner.    -   Children with the juvenile form of MLD (onset between 3-10 years        of age) usually begin with impaired school performance, mental        deterioration, and dementia and then develop symptoms similar to        the late infantile form but with slower progression. Age of        death is variable, but normally within 10 to 15 years of symptom        onset.    -   The adult form commonly begins after age 16 as a psychiatric        disorder or progressive dementia. Adult-onset MLD progresses        more slowly than the late infantile and juvenile forms, with a        protracted course of a decade or more.

In rare cases the body can compensate for the deficiency and the personwill exhibit no symptoms.

There is no cure for MLD, and no standard treatment, it is a terminalillness. Children with advanced juvenile or adult onset, and lateinfantile patients displaying symptoms have treatment limited to painand symptom management. Presymptomatic late infantile MLD patients, aswell as those with juvenile or adult MLD that are either presymptomaticor displaying mild to moderate symptoms, have the option of bone marrowtransplantation (including stem cell transplantation), which is underinvestigation.

The present invention is in one embodiment related to a method fortreating Metachromatic leukodystrophy, by modulation of arylsulfatase Aenzyme activity.

Saposin-Deficiency

In both humans and mice, prosaposin/saposin deficiencies lead to severeneurological deficits.

Human patients with point mutations in the saposin A, B and C showphenotypes of Krabbe disease, metachromatic leukodystrophy and Gaucherdisease, indicating that their primary in vivo substrates aregalactosylceramide, sulfatide and glucosylceramide, respectively.

Krabbe disease, atypical, due to saposin A deficiency: An inheritedbiochemical disorder which results in neurological regression within afew months of birth. Death usually occurs during the first few years oflife. The disorder is similar to Krabbe disease but is differentiated bythe genetic origin of the biochemical defect. Krabbe disease involves adefect in the galactocerebrosidase gene whereas atypical Krabbe diseaseinvolves a defect in the prosaposin gene which causes a deficiency ofsaposin A.

Saposin B, previously known as SAP-1 and sulfatide activator, stimulatesthe hydrolysis of a wide variety of substrates including cerebrosidesulfate, GM1 ganglioside, and globotriaosylceramide by arylsulfatase A,acid beta-galactosidase, and alpha-galactosidase, respectively. Humansaposin B deficiency, transmitted as an autosomal recessive trait,results in tissue accumulation of cerebroside sulfate and a clinicalpicture resembling metachromatic leukodystrophy (activator-deficientmetachromatic leukodystrophy) although with normal arylsulfataseactivity. Saposin B deficiency is a heterogeneous disease with aspectrum similar to that in metachromatic leukodystrophy.

Saposin (SAP-) C is required for glucosylceramide degradation, and itsdeficiency results in a variant form of Gaucher disease;non-neuronopathic Gaucher disease due to SAP-C deficiency. Very highlevels of chitotriosidase activity, chemokine CCL18, and increasedconcentration of glucosylceramide in plasma and normal β-glucosidaseactivity in skin fibroblasts are observed in the patients. A missensemutation, p.L349P, located in the SAP-C domain and another mutation,p.M1L, located in the initiation codon of the prosaposin precursorprotein has been identified.

In a few non-neuronopathic Gaucher patients, a mutation in both SaposinC and saposin D has been identified.

Combined saposin C and D deficiencies in mice lead to a neuronopathicphenotype with glucosylceramide and alpha-hydroxy ceramide accumulation.

In mice, saposin D deficiency is associated with ceramide accumulation,partial loss of Purkinje cells and impaired urinary system function.This phenotype does not mimic the embryonic lethality exhibited by micewith complete deficiency of acid ceramidase, saposin D's cognate enzyme

Two mutations are known in humans that result in complete inactivationof all four saposins and prosaposin. Total saposin deficiency is adevastating disease with involvement of multiple organs and multiplesphingolipids. Combined saposin deficiency (or prosaposin deficiency)has been reported in a case presenting with a severe neurovisceraldystrophy which caused death as a neonate. Multiple sphingolipids wereelevated in the urine, with globotriaosylceramide showing the greatestincrease. A novel mutation in the PSAP gene was identified, beinghomozygous for a splice-acceptor site mutation two bases upstream ofexon 10. This mutation led to a premature stop codon and yielded lowlevels of transcript.

The present invention is in one embodiment related to a method fortreating saposin-deficiency. Said saposin-deficiency may be selectedfrom the group consisting of saposin A deficiency, saposin B deficiency,saposin C deficiency, saposin C deficiency, and combined saposindeficiency (or prosaposin deficiency).

Residual Enzymatic Activity

The lysosomal storage diseases are, as outlined herein above, caused bya defective enzyme. Said defective enzyme may have no residual activity,or may have some residual activity.

Residual enzymatic activity as used herein means that although theenzyme is defective, for example caused by a mutation, the activity ofthe enzyme is not completely abolished, but rather reduced to apathological level.

The present invention relates in one aspect to a bioactive agent for usein treatment of a lysosomal storage disease, and a method for treatmentof an individual with a lysosomal storage disease.

In an embodiment of the present invention, the lysosomal storage diseasewhich is treated according to the present invention is characterised ashaving residual enzymatic activity of the defective enzyme involved inthe disease pathology.

In one embodiment, said residual enzymatic activity is in the range offrom 0.1% to 50%, such as in the range of 0.1 to 1%, for example 1 to2%, such as 2 to 3%, for example 3 to 4%, such as 4 to 5%, for example 5to 6%, such as 6 to 7%, for example 7 to 8%, such as 8 to 9%, forexample 9 to 10%, such as 10 to 11%, for example 11 to 12%, such as 12to 13%, for example 13 to 14%, such as 14 to 15%, for example 15 to 20%,such as 20 to 25%, for example 25 to 30%, such as 30 to 35%, for example35 to 40%, such as 40 to 45%, for example in the range of 45 to 50%residual enzymatic activity.

Current Treatment Modalities for LSD

There are no cures for the lysosomal storage diseases and treatment ismostly symptomatic, although bone marrow transplantation and enzymereplacement therapy (ERT) have been tried with some success. Inaddition, umbilical cord blood transplantation is being performed atspecialized centers for a number of these diseases. Transplantationtherapy is however accompanied by major side effects and often posescomplications to the patients. In addition, substrate reduction therapy,a method used to decrease the accumulation of storage material, iscurrently being evaluated for some of these diseases.

For most of the lysosomal storage diseases, a major unmet need forproviding an effective treatment modality remains.

Enzyme replacement therapy has been developed for a subset of thelysosomal storage diseases, and Cerezyme® has been on the market for anumber of years for the treatment of Gaucher disease. The defectiveenzyme, glucocerebrosidase, is made by recombinant techniques, and givenby intravenous infusion over a few hours. Treatment is not a cure andpatients require lifelong treatment to halt disease progression. Somesymptoms may improve by ERT.

However, for most LSDs, an efficient ERT has not been developed. Thismay be because the production of active enzyme has proven a difficulttask, due to the complex sub-unit structure of the defective enzymes.Indeed, enzymes may fold incorrectly upon production.

For those LSDs in which ERT is available, there are drawbacks which makethis form of therapy less desirable. First and foremost, ERT is a veryexpensive form of therapy, which is a financial burden to the societyand makes it inaccessible to some patients. Also, ERT is targetedspecifically at one disease only. Some side effects has been reportedfor Cerezyme®, including the development of an immune response, nausea,vomiting, abdominal pain, diarrhea, rash, fatigue, headache, fever,dizziness, chills, backache, and rapid heart rate as well as symptomssuggestive of allergic reactions.

The disclosures made in the present invention thus provide a new andinnovative method for treatment of the lysosomal storage diseases. Thisis particularly relevant for these diseases for which no effectivetherapy has been developed, those that may benefit from a less expensivetreatment, and those that may benefit from a combination therapycomprising the bioactive agent of the present invention.

As disclosed herein, the method according to the present inventionprovides for a treatment modality which is substantially cheaper toproduce than ERT and which targets more than one specific lysosomalstorage disorder.

The molecular chaperones, or heat shock proteins, are introduced hereinbelow as the inventors have found that an interaction between heat shockprotein 70 and lysosomal BMP, as introduced herein above, forms thebasis for modulating lysosomal enzymatic activity, and treatinglysosomal storage disorders, according to the present invention.

The Molecular Chaperones

Having spent vast amounts of energy upon first transcribing and thentranslating the genetic code of DNA, the cell has finally produced apolypeptide, whose function presumably is required at this point in thecell's life. However, some final obstacles has to be overcome in orderto achieve a fully functional protein—one of these being correct foldingof this nascent polypeptide chain. The evolutionary imperatives ofachieving correct folding are obvious—not only would it be a terriblewaste of energy to have synthesized a peptide without the properconformation and hence function, but also the aggregation of suchproteins in the cellular lumen could prove detrimental to the cell. Thisaggregation is in fact a very likely outcome, considering theintracellular environment of high protein concentration, so it comes asno surprise that a complicated and sophisticated machinery of proteinsexists to assist protein folding, allowing the functional state ofproteins to be maintained under such conditions. These proteins arecollectively called molecular chaperones, because, like their humancounterparts, they prevent unwanted interactions between their immatureclients.

The molecular chaperones are found in all compartments of a cell whereconformational rearrangements of proteins occur, and although proteinsynthesis is the major source of unfolded peptides in the cell, achallenge to the cell by high temperature or other stimuli that mightrender proteins structurally labile, and hence prone to unfolding andaggregation, is met with a specific cellular response involving theproduction of protective proteins. This response is a phenomenonobserved in every cell type ranging from prokaryotes to eukaryotes andis referred to as the heat-shock- or stress-response. The proteinsinduced by this response are known as the heat shock proteins (HSPs), ofwhich there exist several families. These families are composed of bothsequentially, structurally and functionally related proteins, whereaschaperones from different families can differ markedly both in structureas well as cellular function. A primary example of a family ofchaperones are the Hsp70 proteins, which constitute the central part ofan ubiquitous chaperone system present in most compartments ofeukaryotic cells, in eubacteria, and in many archae. This family hasrecently been implicated in other aspects of cellular homeostasisbesides serving as a chaperone—most markedly through its anti-apoptoticfeatures, its functions in immunity, and the apparent dependence ofcancer cells on the upregulation of Hsp70.

The Heat Shock Protein 70 Family

Hsp70 proteins are involved in a wide range of cellular processesincluding protein folding and degradation of unstable cellular proteinsas well as serving other cytoprotective roles. The common function ofHsp70 in these processes appears to be the binding of short hydrophobicsegments in partially folded polypeptides, thereby facilitating properfolding and preventing aggregation. In eukaryotes, Hsp70 chaperonesinteract in vivo with different classes of proteins that serve toregulate critical steps of their functional cycle; amongst these theJ-domain family protein Hsp40. Furthermore, additional partner proteinshave been identified, some of which are linking Hsp70 to other chaperonesystems such as the Hsp90 system.

Members of the Human Hsp70 Family

Some of the important functions attributed to the molecular chaperonesinclude import of proteins into cellular compartments, folding ofproteins in the cytosol, endoplasmic reticulum and mitochondria,prevention of protein aggregation and refolding of misfolded proteins.At present the human Hsp70 family includes 10 members encoded bydifferent genes, and this section is meant to provide an overview ofthese family members with respect to function, expression patterns andhomology. Some confusion exists about the nomenclature of the differenthuman Hsp70 family members, although a set of general guidelines hasbeen set forth by Tavaria et al., which provides a logical link betweenlocus names, genes and proteins. However, as there still exists someinterspecies confusion, the Hsp70 genes and proteins are referred toherein by their locus name. The name Hsp70 may refer to the twoinducible Hsp70 family members with loci names HSPA1A and HSPA1B or tothe whole Hsp70 family in general as evident from the consensus of thetext. However, as used throughout the present invention, Hsp70 is meantto denote any of the two inducible Hsp70 family members with loci namesHSPA1A and HSPA1B.

HspA1A and HspA1B

The genes transcribed from the loci HSPA1A and HSPA1B are the twoheat/stress-inducible Hsp70-genes and the majority of the literatureconcerning human Hsp70 refers to the proteins encoded by these twogenes. The genes give rise to proteins consisting of 641 amino acids,having 99% homology to each other and were the first human Hsp70 familymembers to be cloned and characterized. The genes are linked in theMHC-class III complex at 6p21.3, are intron-less and with promoterregions containing HSEs, enabling them to bind HSFs and inducetranscription in response to a variety of cellular assaults.

The protein sequence for Homo sapiens heat shock 70 kDaprotein 1A (HSPA1A) is (SEQ ID NO: 1) (Accession no. NM_005345.5):MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVDThe nucleic acid (DNA) sequence for Homo sapiens heat shock 70kDa protein 1A (HSPA1A) is (SEQ ID NO: 2) (Accession no. NM_005345.5):   1 ataaaagccc aggggcaagc ggtccggata acggctagcc tgaggagctg ctgcgacagt  61 ccactacctt tttcgagagt gactcccgtt gtcccaaggc ttcccagagc gaacctgtgc 121 ggctgcaggc accggcgcgt cgagtttccg gcgtccggaa ggaccgagct cttctcgcgg 181 atccagtgtt ccgtttccag cccccaatct cagagcggag ccgacagaga gcagggaacc 241 ggcatggcca aagccgcggc gatcggcatc gacctgggca ccacctactc ctgcgtgggg 301 gtgttccaac acggcaaggt ggagatcatc gccaacgacc agggcaaccg caccaccccc 361 agctacgtgg ccttcacgga caccgagcgg ctcatcggga atgcggccaa aaaccaagtg 421 gcgctgaacc cgcagaacac cgtgtttgac gcgaagcggc tgattggccg caagttcggc 481 gacccggtgg tgcagtcgga catgaagcac tggcctttcc aggtgatcaa cgacggagac 541 aagcccaagg tgcaggtgag ctacaagggg gagaccaagg cattctaccc cgagaagatc 601 tcgtccatgg tgctgaccaa gatgaaggag atcgccgagg cgtacctggg ctacccggtg 661 accaacgcgg tgatcaccgt gccggcctac ttcaacgact cgcagcgcca ggccaccaag 721 gatgcgggtg tgatcgcggg gctcaacgtg ctgcggatca tcaacgagcc cacggccgcc 781 gccatcgcct acggcctgga cagaacgggc aagggggagc gcaacgtgct catctttgac 841 ctgggcgggg gcaccttcga cgtgtccatc ctgacgatcg acgacggcat cttcgaggtg 901 aaggccacgg ccggggacac ccacctgggt ggggaggact ttgacaacag gctggtgaac 961 cacttcgtgg aggagttcaa gagaaaacac aagaaggaca tcagccagaa caagcgagcc1021 gtgaggcggc tgcgcaccgc ctgcgagagg gccaagagga ccctgtcgtc cagcacccag1081 gccagcctgg agatcgactc cctgtttgaa ggcatcgact tctacacgtc catcaccagg1141 gcgaggttcg aggagctgtg ctccgacctg ttccgaagca ccctggagcc cgtggagaag1201 gctctgcgcg acgccaagct ggacaaggcc cagattcacg acctggtcct ggtcgggggc1261 tccacccgca tccccaaggt gcagaagctg ctacaggact tcttcaacgg gcgcgacctg1321 aacaagagca tcaaccccga cgaggctgtg gcctacgggg cggcggtgca ggcggccatc1381 ctgatggggg acaagtccga gaacgtgcag gacctgctgc tgctggacgt ggctcccctg1441 tcgctggggc tggagacggc cggaggcgtg atgactgccc tgatcaagcg caactccacc1501 atccccacca agcagacgca gatcttcacc acctactccg acaaccaacc cggggtgctg1561 atccaggtgt acgaaggcga gagggccatg acgaaagaca acaatctgtt ggggcgcttc1621 gagctgagcg gcatccctcc ggcccccagg ggcgtgcccc agatcgaggt gaccttcgac1681 atcgatgcca acggcatcct gaacgtcacg gccacggaca agagcaccgg caaggccaac1741 aagatcacca tcaccaacga caagggccgc ctgagcaagg aggagatcga gcacatggtg1801 caggaggcgg agaagtacaa agcggaggac gaggtgcagc gcgagagggt gtcagccaag1861 aacgccctgg agtcctacgc cttcaacatg aagagcgccg tggaggatga ggggctcaag1921 ggcaagatca gcgaggcgga caagaagaag gtgctggaca agtgtcaaga ggtcatctcg1981 tggctggacg ccaacacctt ggccgagaag gacgagtttg agcacaagag gaaggagctg2041 gagcaggtgt gtaaccccat catcagcgga ctgtaccagg gtgccggtgg tcccgggcct2101 gggggcttcg gggctcaggg tcccaaggga gggtctgggt caggccccac cattgaggag2161 gtagattagg ggcctttcca agattgctgt ttttgttttg gagcttcaag actttgcatt2221 tcctagtatt tctgtttgtc agttctcaat ttcctgtgtt tgcaatgttg aaattttttg2281 gtgaagtact gaacttgctt tttttccggt ttctacatgc agagatgaat ttatactgcc2341 atcttacgac tatttcttct ttttaataca cttaactcag gccatttttt aagttggtta2401 cttcaaagta aataaacttt aaaattcaaa aaaaaaaaaa aaaaaThe protein sequence for Homo sapiens heat shock 70 kDa protein 1B(HSPA1B) is (SEQ ID NO: 3) (Accession no: NM_005346):MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVDThe nucleic acid (DNA) sequence for Homo sapiens heat shock 70 kDaprotein 1B (HSPA1B) is (SEQ ID NO: 4) (Accession no: NM_005346):   1 ggaaaacggc cagcctgagg agctgctgcg agggtccgct tcgtctttcg agagtgactc  61 ccgcggtccc aaggctttcc agagcgaacc tgtgcggctg caggcaccgg cgtgttgagt 121 ttccggcgtt ccgaaggact gagctcttgt cgcggatccc gtccgccgtt tccagccccc 181 agtctcagag cggagcccac agagcagggc accggcatgg ccaaagccgc ggcgatcggc 241 atcgacctgg gcaccaccta ctcctgcgtg ggggtgttcc aacacggcaa ggtggagatc 301 atcgccaacg accagggcaa ccgcaccacc cccagctacg tggccttcac ggacaccgag 361 cggctcatcg gggatgcggc caagaaccag gtggcgctga acccgcagaa caccgtgttt 421 gacgcgaagc ggctgatcgg ccgcaagttc ggcgacccgg tggtgcagtc ggacatgaag 481 cactggcctt tccaggtgat caacgacgga gacaagccca aggtgcaggt gagctacaag 541 ggggagacca aggcattcta ccccgaggag atctcgtcca tggtgctgac caagatgaag 601 gagatcgccg aggcgtacct gggctacccg gtgaccaacg cggtgatcac cgtgccggcc 661 tacttcaacg actcgcagcg ccaggccacc aaggatgcgg gtgtgatcgc ggggctcaac 721 gtgctgcgga tcatcaacga gcccacggcc gccgccatcg cctacggcct ggacagaacg 781 ggcaaggggg agcgcaacgt gctcatcttt gacctgggcg ggggcacctt cgacgtgtcc 841 atcctgacga tcgacgacgg catcttcgag gtgaaggcca cggccgggga cacccacctg 901 ggtggggagg actttgacaa caggctggtg aaccacttcg tggaggagtt caagagaaaa 961 cacaagaagg acatcagcca gaacaagcga gccgtgaagc ggctgcgcac cgcctgcgag1021 agggccaaga ggaccctgtc gtccagcacc caggccagcc tggagatcga ctccctgttt1081 gagggcatcg acttctacac gtccatcacc agggcgaggt tcgaggagct gtgctccgac1141 ctgttccgaa gcaccctgga gcccgtggag aaggctctgc gcgacgccaa gctggacaag1201 gcccagattc acgacctggt cctggtcggg ggctccaccc gcatccccaa ggtgcagaag1261 ctgctgcagg acttcttcaa cgggcgcgac ctgaacaaga gcatcaaccc cgacgaggct1321 gtggcctacg gggcggcggt gcaggcggcc atcctgatgg gggacaagtc cgagaacgtg1381 caggacctgc tgctgctgga cgtggctccc ctgtcgctgg ggctggagac ggccggaggc1441 gtgatgactg ccctgatcaa gcgcaactcc accatcccca ccaagcagac gcagatcttc1501 accacctact ccgacaacca acccggggtg ctgatccagg tgtacgaggg cgagagggcc1561 atgacgaaag acaacaatct gttggggcgc ttcgagctga gcggcatccc tccggccccc1621 aggggcgtgc cccagatcga ggtgaccttc gacatcgatg ccaacggcat cctgaacgtc1681 acggccacgg acaagagcac cggcaaggcc aacaagatca ccatcaccaa cgacaagggc1741 cgcctgagca aggaggagat cgagcgcatg gtgcagaagg cggagaagta caaagcggag1801 gacgaggtgc agcgcgagag ggtgtcagcc aagaacgccc tggagtccta cgccttcaac1861 atgaagagcg ccgtggagga tgaggggctc aagggcaaga tcagcgaggc ggacaagaag1921 aaggttctgg acaagtgtca agaggtcatc tcgtggctgg acgccaacac cttggccgag1981 aaggacgagt ttgagcacaa gaggaaggag ctggagcagg tgtgtaaccc catcatcagc2041 ggactgtacc agggtgccgg tggtcccggg cctggcggct tcggggctca gggtcccaag2101 ggagggtctg ggtcaggccc taccattgag gaggtggatt aggggccttt gttctttagt2161 atgtttgtct ttgaggtgga ctgttgggac tcaaggactt tgctgctgtt ttcctatgtc2221 atttctgctt cagctctttg ctgcttcact tctttgtaaa gttgtaacct gatggtaatt2281 agctggcttc attatttttg tagtacaacc gatatgttca ttagaattct ttgcatttaa2341 tgttgatact gtaagggtgt ttcgttccct ttaaatgaat caacactgcc accttctgta2401 cgagtttgtt tgtttttttt tttttttttt ttttttgctt ggcgaaaaca ctacaaaggc2461 tgggaatgta tgtttttata atttgtttat ttaaatatga aaaataaaat gttaaacttt2521 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

HspA1L and HspA2

Two Hsp70 family members have been termed “chauvinist genes” becausemale germ cells favor their expression with strong prejudice. The hspA1Lgene is a constitutively expressed intron-less Hsp70 family memberlocated 4 kb telomeric to the HSPA1A locus in the same MHC-class IIIcomplex on chromosome 6. It is expressed in low amounts both before andafter heat shock but with the expression pattern favoring the testes inmouse, rat and humans with the 641 amino acids (aa) protein being 90%homologous to HspA1A. The hspA2 gene was first isolated from a mousegenomic library and has later been shown to be constitutively expressedalbeit in low levels in various tissues in the human body includingskeletal muscle, ovary, small intestine, colon, brain, placenta and thekidneys, but highly expressed in testis. Its expression, or rather lackthereof, has been connected with abnormal human spermatogenesis and malehspA2^((−/−))mice are sterile. The gene is located on chromosome 14,giving rise to a 639 aa protein with 84% homology to HspA1A, althoughthe exact location is subject to discussion as two papers have presenteddifferent loci positions—14q24.1 vs. 14q22.

HspA6 and HspA7

The hspA6 and hspA7 genes are heat inducible members of the Hsp70 familywith no apparent counterparts in mice. They contain HSEs in theirpromoter-sites and the genes are intron-less. They are co-localized onchromosome 1 and are 94% homologous to each other in the nucleotidesequence. However, only HspA6 is functional as the hspA7 gene harbors asingle nucleotide insertion generating a premature stop codon at +1324.The HspA6 protein is 643 as long and shows 77% homology to HspA1A andHspA1B.

HspA5 and HspA9

The hspA5 and hspA9 genes are the two compartment-specific members ofthe Hsp70 family. The 655 aa HspA5 protein is located in the endoplasmicreticulum (ER) and facilitates folding and transport of newlysynthesized proteins in this compartment. The protein is 64% homologousto HspA1A, the gene being located at 9q34. The 679 aa HspA9 protein islocated in the mitochondria where it assists in folding of proteinsafter their transport across the mitochondrial membrane. HspA9 islocated at 5q31.1, the protein being 52% homologous to HspA1A.

HspA8

The cognate Hsp70 member known as Hsc70 is encoded by a gene named hspA8at 11q24, giving rise to a 646 aa protein with 86% homology to HspA1A,and is constitutively expressed in all tissues and cell lines. Theprotein is analogous to Hsp70 in its cellular functions, providing therequired chaperoning under normal circumstances, but has also beenascribed a role in the un-coating of clathrin-coated vesicles as well asin chaperone-mediated autophagy.

HspA3 and HspA4

These will not be discussed here, as there is doubt as to whether HSPA3exists at all and since HSPA4 is most likely a member of the Hsp110family and nothing is known about it so far, except for its chromosomallocation at 5q31.1-2.

TABLE 1 List of the Human Hsp70 Gene Family. Name Used % aa herein,Homology Locus Gene/Protein Position to HSPA1A Alternative Names HSPA1AhspA1A/HspA1A 6p23.1 100  Hsp70; Hsp72; Hsp70-1 (Hsp70) HSPA1BhspA1B/HspA1B 6p23.1 99 Hsp70; Hsp72; Hsp70-2 (Hsp70) HSPA1LhspA1L/HspA1L 6p23.1 90 Hsp70-Hom; Hsp70t HSPA2 hspA2/HspA2 14q24.1 84Hsp70-3 HSPA4 hspA4/HspA4 5q31.1 31 Hsp70RY; APG-2 HSPA5 hspA5/HspA59q34 64 BiP; GRP78 HSPA6 hspA6/HspA6 1q 84 Hsp70-6; Hsp70B′ HSPA7hspA7/HspA7 1q — Hsp70-7; Hsp70B HSPA8 hspA8/HspA8 11q24 86 Hsc70; Hsp73(Hsc70) HSPA9 hspA9/HspA9 5q31.1 52 GRP75; PBP74; mtHsp75; mortalin;mot-2 The genes are listed according to locus name, names used herein,chromosomal location (position), amino acid homology to HspA1A as wellas alternative names often seen in the literature.

Transcriptional Regulation of Hsp70

Genomic foot printing of the human Hsp70 promoter has revealed that heatshock/stress induces a rapid binding of heat shock transcription factors(HSF) to a region encompassing nGAAn sequences named heat shock elements(HSEs). Under normal conditions Hsp70 is bound to HSFs, which reside inthe cytosol, but during stress the HSFs are separated from Hsp70 andadapt a homotrimeric conformation upon phosphorylation by PKC or otherserine/threonine kinases. The HSF trimers enter the nucleus, where theybind HSEs located in the promoter region of Hsp70 genes and becomefurther phosphorylated by HSF kinases.

Three HSFs have so far been characterized in humans (HSF1, HSF2 andHSF4). HSF1 is the major transcription factor activated under moststress conditions and responds to a wide range of stimuli, which can becategorized into physiological (e.g. cell division, hormonalstimulation), pathological (e.g. infections, fever, inflammation,malignancy) and environmental conditions (e.g. heat shock, heavy metals,ethanol). HSF2 responds only to hemin, whereas HSF4 is preferentiallyexpressed in the human heart, pancreas, brain and skeletal muscle, lacksthe c-terminal hydrophobic repeat that is shared among all vertebrateHSFs and appears to repress expression of HSPs. The Hsp70 generegulation responsible for synthesis of the constitutively expressedHsp70 (Hsc70) is not clearly understood, but HSFs do not seem to beinvolved.

Although the HSFs are the most prominent of the factors regulating HSPexpression, other transcription factors have been shown to possess thesame capability. Specific CCAAT-box binding factors (CBF) have beenshown to induce Hsp70 transcription, the tumor-suppressor p53 canrepress transcription by binding to the promoter-region of Hsp70 and byneutralizing CBF, and HSFs can be antagonized by the heat shock factorbinding protein 1 (HSBP1), which in this way attenuates Hsp70transcription.

Structural and Functional Properties of Hsp70

The structure and function of the Hsp70 system are best understood forthe eubacterial Hsp70, DnaK, its Hsp40 co-chaperone DnaJ and thenucleotide exchange factor GrpE.

However, the mechanism is generally considered to be analogous ineukaryotes, although evidence suggests an uncoupling of GrpE. Thissection will focus on the eukaryotic Hsp70 system, but will also includecomments on the eubacterial system, where this is consideredappropriate.

Hsp70 is comprised of two functional entities—an N-terminal ATPasedomain and a smaller C-terminal peptide-binding domain. The ATPasedomain is comprised of two subdomains separated by a cleft containingthe nucleotide-binding site, which determines the peptide-bindingproperties of the C-terminal domain. When ATP is bound, peptidesubstrates bind and dissociate rapidly, albeit with low affinity,whereas in a state where either no nucleotide or ADP is bound to theN-terminal domain, the rates of peptide binding and dissociationdecrease and the affinity increases. ATP hydrolysis thus serves as amolecular switch between two states of Hsp70, the cycling of which isregulated by the J-domain family protein Hsp40 in eukaryotes and DnaJand GrpE in eubacteria. The N-terminal J-domain of Hsp40 binds to Hsp70accelerating ATP-hydrolysis, hereby facilitating peptide capture,whereas the C-terminal part of Hsp40 functions as a chaperone byrecognizing hydrophobic peptides, whereby Hsp70 is recruited to nascentpolypeptide chains. It is important to note that the molecularchaperones do not provide specific steric information for the folding ofthe bound protein, but rather inhibit unproductive interactions, thusallowing the protein to fold more efficiently into its native structure.

In eubacteria, GrpE induces the release of ADP from DnaK (bacterialHsp70), whereas for eukaryotic Hsp70 proteins such a factor appears tobe dispensable because the rate-limiting step in this ATPase cycle isnot the dissociation of bound ADP but rather the ATP-hydrolysis itself.However, additional proteins serve to regulate Hsp70 function ineukaryotes; the homo-oligomeric protein Hip (Hsp70 interacting protein)serving as a positive regulator by stabilizing the ADP-bound state ofHsp70, whereas the proteins Carboxy-terminus of Hsp70-binding protein(CHIP) and Bcl-2-associated athanogene-1 (Bag-1) both have inhibitoryeffects—CHIP by inhibiting the ATPase activity of Hsp70 and Bag-1 byantagonizing the refolding activity of Hsp70. Further interactions areprovided by the two human Hsp40 proteins Hdj1 and Hdj2, which, besidestheir Hsp40 functions (described above), have been shown to facilitatethe coupling of Hsp70 and Hsp90 through Hop (Hsp-organizing protein), anadaptor protein which physically links the chaperones through its twotetratricopeptide repeat (TPR) domains that bind the extended C-terminalsequences of Hsp70 and Hsp90, respectively. It has recently been shownthat some of the above mentioned proteins are regulatory in the transferof non-native or irreversible misfolded proteins from the chaperones tothe ubiquitin-proteasome machinery. The protein CHIP is, apart from itsnegative regulatory role on Hsp70, able to associate with Hsp90 throughan N-terminal TPR domain and targets Hsp90 substrates for degradationthrough a C-terminal ubiquitin ligase domain, but is also capable ofcooperating functionally with BAG-1, which binds to Hsp70 (as well asthe proteasome. These findings provide a possible link between themechanisms that integrate chaperone-assisted folding and proteolyticdegradation, the two main components of protein quality control in thecytosol.

Cytoprotection Via Hsp70

Apart from its anti-apoptotic abilities as a consequence of being amolecular chaperone, i.e. facilitating protein folding under otherwisedenaturing conditions, Hsp70 is also able to affect the survival ofcells in various other ways, including protection of mitochondrialfunction after ischemia-reperfusion injury, blocking activation of thestress kinase c-jun N-terminal kinase (JNK) upon stimulation of primaryfibroblasts with TNF, and a Hsp70/Bag-1 complex has been proposed toregulate cell growth and mitogenesis during conditions of cellularstress. The ability of Hsp70 to protect cells from cell death induced byan array of stimuli such as TNF, TRAIL, oxidative stress, UV-radiationand the anti-cancer drugs doxorubicin, etoposide and taxol furtheremphasize its anti-apoptotic features. Finally, reports have alsoprovided evidence of more direct interactions between Hsp70 and theapoptotic machinery as Hsp70 has been shown to antagonizeapoptosis-inducing factor (AIF), as well as exert an anti-apoptoticfunction downstream of caspase-3.

Recent evidence also suggests that parts of the potent cytoprotectiveeffect of Hsp70 are due to stabilization of lysosomal membranes. Inevidence of this, the depletion of Hsp70 triggers an earlypermeabilization of lysosomal membranes and cathepsin-mediated celldeath in cancer cells, and exogenous Hsp70 effectively inhibitslysosomal destabilization induced by various stresses. Furthermore, micedeficient for Hsp70 suffer from pancreatitis caused by the leakage oflysosomal proteases into the cytosol. All of these events stress therole of Hsp70 as an important regulator of PCD and hence survival factorfor cells.

Hsp70 in Cancer

Hsp70 is often over-expressed in malignant human tumors, and itsexpression correlates with poor prognosis in breast and endometrialtumors. In line with this, Hsp70 increases the tumorigenic potential ofrodent cells implanted into immuno-compromised or syngeneic animals.

The role of Hsp70 as an essential factor for cancer cell survival isfurther substantiated from a report by Wei et al., who made the firstdepletion-study of Hsp70 in cancer cells. The results indicated thatwhen Hsp70 expression was inhibited in various cancer cell lines by theuse of an antisense-oligomer, inhibition of cell proliferation andsubsequent apoptosis was induced. This work has been substantiated in aseries of experiments in which adenoviral antisense-mediated depletionof Hsp70 triggers a tumor cell-specific lysosomal death program.

In vivo studies utilizing orthotopic xenografts of glioblastoma andbreast carcinomas as well as sub-cutaneous xenografts of colon-carcinomain immunodeficient mice has further demonstrated the anti-cancerpotential of Hsp70 depletion, as the tumors of mice receivinglocoregional application of the above-mentioned adenoviral constructshowed massive apoptosis-like cell death and recruitment of macrophages.These studies clearly demonstrate the dependence of some tumors upon thepresence of Hsp70, although other studies have argued that thecytotoxicity observed in cell culture upon adenovirus-mediated depletionof Hsp70 is due to a combination of virally mediated cell-stress andHsp70-downregulation. Despite this controversy, the cytotoxicity in cellculture induced by the depletion of Hsp70 was not dependent on caspasessince neither overexpression of Bcl-2 nor pharmacological inhibition ofcaspases could rescue the cells. Rather, the triggering of LMP andrelease of cathepsins to the cytosol was the likely death-inducingevents as the inhibition of cysteine cathepsins conferred significantcytoprotection. Furthermore, depletion of Hsp70 in the before mentionedtumor xenografts in mice lead to cathepsin release and tumor cell death.

As mentioned earlier, one of the cytoprotective mechanisms of Hsp70,which many cancer cells seem to have adapted, is the translocation ofHsp70 to the endo-lysosomal compartment where it serves amembrane-protective role. This translocation may not only be driven bythe need to protect the lysosomal membranes, as studies have shown thatmore than 50% of tumors show localization of Hsp70 on theplasma-membrane surface—an area which is directly connected with theendo-lysosomal compartment via endocytosic and secretory events, asdescribed earlier. The surface-exposed Hsp70 presents a unique epitopewhich can act as a recognition structure for natural killer (NK) cells,stimulating their proliferation and cytolytic activity. NK cellsactivated by this Hsp70 peptide sequence has been shown to inhibittumour growth in mice with severe combined immunodeficiency (SCID), apossible mechanism for this could be that the cell-surface-bound Hsp70mediates apoptosis by the specific binding and uptake of granzyme B,independent of perforin.

As previously written, the endo-lysosomal membranes and plasma membranesare constantly interchanged. Thus, the presence of Hsp70 on the surfaceof cancer cells could be an “unfortunate” consequence of two events thatpromote tumor progression; the secretion of cathepsins, which promotesinvasion and angiogenesis, and the localization of Hsp70 on thelysosomal membranes, which prevents accidental release of cysteinecathepsins to the cytosol and ensuing cell death.

Extracellular Hsp70

As evident from the former paragraphs, the intracellular functions ofHsp70 are essential for proper cell homeostasis, not least so in theface of noxious challenges. However, interesting roles are also emergingfor extracellular Hsp70 (eHsp70) especially when it comes to immune andinflammatory responses, which again might have important roles for theclearance of cancer cells. Furthermore, involvement in a generalphysiological adaptation to stress and protection versus cellular damageare also emerging themes for eHsp70.

Extracellular Hsp70 and Neuroprotection

The first evidence for the presence of eHsp70 came from studies in thesquid giant axon, in which it was shown that elevation of temperatureinduced a set of heat shock proteins in the glial sheath surrounding theaxon which where transferred into the axon. These findings where soonreproduced in cultured rat embryo cells, and importantly, already atthis point, evidence was presented for a non-classical pathway ofexocytosis being responsible for the release of Hsp70 as neithermonensin nor colchicine, both inhibitors of the classical secretorypathway, could block the secretion of Hsp70. Since these publications,other reports have provided examples of release of Hsps by glia and theuptake by neurons in various animal model systems such as frogs,crayfish and rats. Support of a role for glia cells as sources of eHsp70in humans was provided by a study of cultured human glioblastoma cells.This study showed that under control conditions the cells released ˜10pg of Hsp70 per million cells to the medium in a time period of 24 h.This release was increased 2.5-5-fold when a 20 min heat shock wasapplied in the beginning of the time period. Importantly, this studyalso showed that the release of eHsp70 was greater than what could beaccounted for by cell death. These data all support the originallysuggested hypothesis set forth by Tytell et al., that glial release ofHsps may be a way to support neuron function during metabolic stress.

In vivo evidence for eHsp70 having a neuroprotective role during acutestress comes from a variety of studies. A study by Tidwell et al. foundthat eHsp70 is capable of reducing the amount of post-axotomy motorneuron cell death, when eHsp70 was applied via a gel-sponge afteraxotomy. In the same study, increased survival of dorsal root ganglionsensory neuron cells where also observed upon Hsp70 administration,albeit this depended on slightly higher doses of Hsp70 than the motorneurons. In addition, eHsp70 has been shown to protect motor neuronsotherwise destined to die during chick embryonic development, and alsoprotect motor neurons isolated from chick spinal cords upon trophicfactor deprivation. An in vivo protective role for eHsp70 has also beendescribed when it comes to light damage of the retina. In this study, Yuet al., intravitreally injected a solution of recombinant Hsp70 andHsc70 after exposure to damage-inducing light at a dose which hadpreviously been described to cause extensive photoreceptor degeneration.Interestingly, the presence of the eHsp70 mixture in the vitreouschamber of the right eye resulted in significantly more photoreceptorssurviving in the retina. Furthermore, evaluation of uptake offluorescein-labelled Hsc/Hsp70 demonstrated that it was present in theretina 6 h after administration. Extracellular Hsp70 administered viaintranasal treatment has also been shown to prevent the consequences ofunavoidable stress in rats and it was recently described thatintraperitoneally injected recombinant human Hsp70 was effective inincreasing the lifespan, delaying symptom onset, preserving motorfunction and prolonging motor neuron survival in a mouse model ofamyotrophic lateral sclerosis. Additional in vitro work using Hsp70 orthe Hsc/Hsp70 mixture in neuronal systems has furthermore shown thateHsp70 can enhance neuronal cell stress tolerance and reducepolyglutamine toxicity and aggregation.

Extracellular Hsp70 and Immunity

Beside roles in cytoprotection, both plasma membrane-associated as wellas free systemic eHsp70 have been documented to serve roles in immunity.Considering that one of the major functions of Hsp70 is to chaperoneintracellular proteins, it is perhaps not surprising that it can beinvolved in binding of immunogenic peptides and assist in thepresentation of these by major histocompatibility complex (MHC) class 1molecules. Furthermore, tumor-derived eHsp70 has been shown to chaperoneimmunogenic peptides and selectively bind to antigen presenting cells(APC). Following receptor-mediated endocytosis these Hsp70-peptidecomplexes are then presented on MHC class 1 molecules leading to acytotoxic t-cell response. In addition to the chaperoning ofself-antigens, Hsp70 is also capable of binding microbial peptides andunmethylated CpG motifs in bacterial DNA.

In addition to its role as an antigen-presenting chaperone, eHsp70 hasalso been implicated in the stimulation of innate immunity. Whilst anumber of cell types have been shown to release Hsp70, eHsp70 has alsobeen shown to bind to a number of receptors on different leucocytesub-populations including natural killer (NK) cells, macrophages,monocytes and dendritic cells. The receptors involved in eHsp70recognition mainly include pattern recognition receptors (PRR's) andconsist of a variety of receptors from different receptor families suchas the toll like receptors (TLR), scavenger receptors and c-typelectins. Upon receptor binding, eHsp70 is capable of eliciting a widecytokine-response including release of pro-inflammatory cytokines suchas TNF-α, IL-1b, IL-12, IL-6 and GM-CSF, a process triggered bytranslocation of NF-kB to the nucleus, suggesting a cytokine action ofeHsp70, which has also led to the suggestion of coining the termchaperokine to eHsp70 in order to better describe the unique functionsof eHsp70 as both a chaperone and cytokine.

Much of the in vivo work on a role of eHsp70 in immunity has beenconducted in rodent models. For example, increases in eHsp70concentration in response to tail-shock were associated with reducedinflammation and quicker recovery times following a sub-cutaneous E.Coli-injection. In addition, in vivo delivery of Hsp70 into miceaccelerated wound closure, a feature which was likely due to enhancedmacrophage phagocytosis of wound debris.

Evidence for the immunomodulatory roles of Hsp70 in humans is lacking,but studies have demonstrated relationships between increased eHsp70 andimproved prognosis/outcome for brain trauma, although the contrary hasalso been shown. However, it is also known that concentrations of eHsp70decline with advancing age, which may be indicative of an age-relatedreduced ability to mount a full stress-response, which again couldaccount for the increased morbidity and mortality seen with ageing,although this remains purely speculative.

Release of Hsp70

Aside for the data demonstrating transfer of eHsp70 between neighboringcells such as in the glia/axon model, several reports have documentedthe presence of free eHsp70 in the circulation. For Hsp70 to be presentin this compartment, it necessarily has to be released from anorgan/cell. Two major ways of achieving this are usually considered. Oneis a passive way in which the observation of eHsp70 in the peripheralcirculation is the consequence of release from an intracellular pool ofHsp70 due to cell lysis or death. Alternatively, or perhapsadditionally, Hsp70 is actively released via a non-classical exocytoticpathway.

It has been suggested that Hsp70 along with other heat shock proteinsare only released under pathological circumstances resulting in necroticdeath and not during programmed cell death. No doubt, severe trauma andpathological conditions resulting in necrosis can lead to the release ofHsp70 to the bloodstream. This has been well documented and would alsologically be expected. Recent studies however, have shown that Hsp70 canbe released from intact cells by active mechanisms and that the degreeof stimulus determines the mode of release. Strong evidence for thenon-necrotic release of Hsp70 also comes from studies onexercise-induced release of eHsp70 to the peripheral bloodstream.Dependent on the mode of exercise (the higher the physical strain, themore release) major increases of eHsp70 can be detected in theperipheral bloodstream, and importantly, no known studies have reporteda direct correlation between eHsp70 and markers of muscle damage. ThateHsp70 can be released regardless of cellular or tissue damage hasfurthermore been elegantly demonstrated by Fleshner and co-workers whohave shown that psychological stress such as predatory fear and electricshock can evoke a stress induced eHsp70 release, a process which wassuggested to be dependent on cathecholamine signaling.

The way by which hsp70 leaves the cell is still unclear though, notleast so because Hsp70 does not contain any classical peptide leadersequence, which could target it for secretion. In addition, as classicalsecretion was already questioned early, this suggests that alternatemechanisms for eHsp70 release must exist. It has been demonstrated thateHsp70 can be released in vesicles characterized as exosomes, butevidence has also been presented that eHsp70 can be released as freeeHsp70, both in cellular systems as well as in vivo. It has beensuggested that lipid rafts are needed for eHsp70 release although thishas also been disputed. Moreover, it has been shown that a functionallysosomal compartment is necessary for release of eHsp70 and that thisrelease is accompanied by the presence of lysosomal marker proteins onthe surface of the cells, suggesting a secretion dependent on plasma-and lysosomal membrane fusion. Regardless of whether the release is viaexosomes or via direct release from lysosomes, it is interesting to notethat some sort of secretory MVB/late endosomal/lysosomal compartment isapparently involved in all modes of release.

As catecholamines via the □₁-adrenergic receptor can lead tointracellular calcium-fluxes, and since the same calcium-fluxes has beensuggested to cause exocytosis of exosomes, multivesicular bodies andlysosomes, a current hypothesis is that under times of stress, increasesin noradrenaline acting upon □₁-adrenergic receptors results in acalcium flux within the cell and a subsequent release of Hsp70 withinexosomes.

Bioactive Agent According to the Present Invention

The present invention relates in one embodiment to the modulation ofenzymatic activity, wherein said enzyme interacts with BMP, by the useof a bioactive agent capable of increasing the intracellularconcentration and/or activity of Hsp70.

The modulation of enzymatic activity according to the present inventioncan be obtained by providing one of the following classes of compoundsand therapies, which increases the intracellular concentration and/oractivity of Hsp70:

-   -   Hsp70, or a functional fragment or variant thereof    -   Hsp70 inducers and co-inducers        -   Small-molecule drugs such as Bimoclomol and Arimoclomol        -   Membrane fluidizers such as benzyl alcohol        -   Sub-lethal heat-therapy (≤42° C.) or hyperthermia        -   Certain drugs from the group of anti-inflammatory and            anti-neoplastic drugs        -   Cellular stress            -   Reactive oxygen species (ROS)            -   Adrenalin, noradrenalin            -   UV light            -   Radiation therapy

A bioactive agent according to the present invention is thus any agent,chemical or compound that increases the intracellular concentrationand/or activity of Hsp70; and includes HSP70 itself, or a functionalfragment or variant thereof, and any Hsp70 inducer or co-inducer knownto the skilled person, whereby said bioactive agent is capable ofmodulating the activity of an enzyme which interacts with BMP.

It follows that a bioactive agent may increase the intracellularconcentration and/or activity of Hsp70 either directly or indirectly.

In one embodiment, the bioactive agent according to the presentinvention is Hsp70, or a functional fragment or variant thereof.

In another embodiment, the bioactive agent according to the presentinvention is an Hsp70 inducer or co-inducer.

In one embodiment, the bioactive agent according to the presentinvention comprises a combination of Hsp70, or a functional fragment orvariant thereof, and an Hsp70 inducer or co-inducer.

It is an aspect of the present invention to provide a bioactive agentcapable of increasing the intracellular concentration and/or activity ofHsp70, for use as a medicament.

It is a further aspect of the present invention to provide a bioactiveagent capable of increasing the intracellular concentration and/oractivity of Hsp70, for use in the treatment of a lysosomal storagedisorder.

It is a further aspect of the present invention to provide a bioactiveagent capable of increasing the intracellular concentration and/oractivity of Hsp70, for use as a medicament or for use in the treatmentof a lysosomal storage disorder.

In one embodiment, said treatment may be prophylactic, curative orameliorating. In one particular embodiment, said treatment isprophylactic. In another embodiment, said treatment is curative. In afurther embodiment, said treatment is ameliorating.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

In a particular embodiment, said lysosomal storage disorder isNiemann-Pick disease type A or B. In another particular embodiment, saidlysosomal storage disorder is Farber disease. In another particularembodiment, said lysosomal storage disorder is Krabbe disease. Inanother particular embodiment, said lysosomal storage disorder isMetachromatic leukodystrophy. In another particular embodiment, saidlysosomal storage disorder is Sialidosis. In another particularembodiment, said lysosomal storage disorder is Fabry disease. In yetanother particular embodiment, said lysosomal storage disorder isGaucher disease. In yet another particular embodiment, said lysosomalstorage disorder is saposin-deficiency.

It is also an aspect of the present invention to provide a bioactiveagent capable of increasing the intracellular concentration and/oractivity of Hsp70, for use in the treatment of a lysosomal storagedisorder, wherein said lysosomal storage disorder is one, such as two,for example three, such as four, for example five, such as six, forexample seven disorders selected from the group consisting ofNiemann-Pick disease, Farber disease, Krabbe disease, Fabry disease,Gaucher disease, Metachromatic leukodystrophy, Sialidosis andsaposin-deficiency.

It follows that the bioactive agent according to the present inventionmay be used for the treatment of a subset of the lysosomal storagedisorders selected from the group consisting of Niemann-Pick disease,Farber disease, Krabbe disease, Fabry disease, Gaucher disease,Metachromatic leukodystrophy, Sialidosis and saposin-deficiency.

In one particular embodiment, the bioactive agent according to thepresent invention may be used for the treatment of Niemann-Pick diseasetype A and B and Farber disease.

In one embodiment, the bioactive agent according to the presentinvention comprises a combination of Hsp70, or a functional fragment orvariant thereof, and a substance which increases the interaction betweenHsp70 and BMP.

It is a still further aspect of the present invention to provide the useof a bioactive agent capable of increasing the intracellularconcentration and/or activity of Hsp70, for the manufacture of amedicament for treatment of a lysosomal storage disorder.

Bioactive Agent—Hsp70, or a Functional Fragment or Variant Thereof

The present invention relates in one embodiment to the modulation ofenzymatic activity, wherein said enzyme interacts with BMP, by the useof Hsp70, or a functional fragment or variant thereof.

It is an aspect of the present invention to provide Hsp70, or afunctional fragment or variant thereof, for use as a medicament.

It is a further aspect of the present invention to provide Hsp70, or afunctional fragment or variant thereof, for use in treating lysosomalstorage disorders.

It is a still further aspect of the present invention to provide the useof Hsp70, or a functional fragment or variant thereof, for themanufacture of a medicament for treating lysosomal storage disorders.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

It is understood that Hsp70, or a functional fragment or variantthereof, according to the present invention may be any natural orsynthetic product, and may be produced by any conventional techniqueknown to the person skilled in the art.

In one embodiment, Hsp70, or a functional fragment or variant thereof,is purified from a natural source. Said natural source may be any plant,animal or bacteria which expresses, or may be induced to express, Hsp70in a form suitable for administering to an individual in need thereof.

In a preferred embodiment however, Hsp70, or a functional fragment orvariant thereof, is made synthetically. It follows that Hsp70, or afunctional fragment or variant thereof, may in one preferred embodimentbe a recombinant protein made by conventional techniques therefore andas such is denoted rHsp70.

The Hsp70 according to the present invention, synthetic or natural, mayhave a sequence which is derived from any suitable species of plant,animal or bacteria. In one embodiment, said rHsp70 is derived from amammal. Said mammal may be selected form the group consisting of human(Homo sapiens), mouse (Mus musculus), cow, dog, rat, ferret, pig, sheep,and monkey. In another embodiment, said rHsp70 is derived from bacteria.

Hsp70 is characterized in part by having a very high degree ofinterspecies sequence conservation, thus possibly allowing for Hsp70derived from one species to be used in another species without elicitinga harmful immune response.

In one particular embodiment, said rHsp70 has a sequence derived fromhuman Hsp70.

In one particular embodiment, said rHsp70 has a sequence derived frommore than one species. Said Hsp70, or a functional fragment or variantthereof, may thus in one embodiment be a chimera.

A recombinant protein is a protein that is derived from recombinant DNA.Recombinant DNA is a form of DNA that does not exist naturally, which iscreated by combining DNA sequences that would not normally occurtogether. In terms of genetic modification, recombinant DNA isintroduced through the addition of relevant DNA into an existingorganismal DNA, such as the plasmids of bacteria, to code for differenttraits for a specific purpose. It differs from genetic recombination, inthat it does not occur through processes within the cell, but isengineered by man.

In one embodiment, the Hsp70 according to the present invention has 100%homology to the wild-type Hsp70 protein. In another embodiment, theHsp70 according to the present invention has less than 100% homology tothe wild-type Hsp70 protein, such as between 99.9 to 95% homology, forexample 95 to 90% homology, such as 90 to 85% homology, for example 85to 80% homology, such as 80 to 75% homology, for example 75 to 60%homology to the wild-type protein. Regardless of the degree of homology,any variant of Hsp70 that retains its ability to modulate the enzymaticactivity of an enzyme which binds to BMP is encompassed by the presentinvention.

In one embodiment, the bioactive agent is Hsp70. In one embodiment, saidHsp70 is full length Hsp70.

It is also an embodiment to provide a functional fragment or variant ofHsp70. As defined herein, a functional fragment or variant is anyfragment of Hsp70 having the desired function, which in terms of thepresent invention is a capability to modulate the enzymatic activity ofan enzyme, wherein said enzyme interacts with BMP.

In one embodiment, the bioactive agent is a functional fragment orvariant of Hsp70.

In one embodiment, the bioactive agent is a functional fragment orvariant of Hsp70, in which Hsp70 is modified by deletion(s), addition(s)or substitution(s) of the wild type Hsp70.

The wild type Hsp70 protein has a total length of 641 amino acids. Afragment of Hsp70 is in one embodiment meant to comprise any fragmentwith a total length of less than the wild type protein of 641 aminoacids, such as less than 625 amino acids, for example less than 600amino aids, such as less than 575 amino acids, for example less than 550amino aids, such as less than 525 amino acids, for example less than 500amino aids, such as less than 475 amino acids, for example less than 450amino aids, such as less than 425 amino acids, for example less than 400amino aids, such as less than 375 amino acids, for example less than 350amino aids, such as less than 325 amino acids, for example less than 300amino aids, such as less than 275 amino acids, for example less than 250amino aids, such as less than 225 amino acids, for example less than 200amino aids, such as less than 175 amino acids, for example less than 150amino aids, such as less than 125 amino acids, for example less than 100amino aids, such as less than 75 amino acids, for example less than 50amino aids, such as less than 25 amino acids.

The wild type Hsp70 protein has a total length of 641 amino acids. Afragment of Hsp70 is in one embodiment meant to comprise any fragmentwith a total length of more than 10 amino acids, such as more than 25amino acids, for example more than 50 amino aids, such as more than 75amino acids, for example more than 100 amino aids, such as more than 125amino acids, for example more than 150 amino aids, such as more than 175amino acids, for example more than 200 amino aids, such as more than 225amino acids, for example more than 250 amino aids, such as more than 275amino acids, for example more than 300 amino aids, such as more than 325amino acids, for example more than 350 amino aids, such as more than 375amino acids, for example more than 400 amino aids, such as more than 425amino acids, for example more than 450 amino aids, such as more than 475amino acids, for example more than 500 amino aids, such as more than 525amino acids, for example more than 550 amino aids, such as more than 575amino acids, for example more than 600 amino aids, such as more than 625amino acids.

It follows that the total length of the fragment of Hsp70 according tothe present invention may in one embodiment be within the range of 5 to25 amino acids, such as 25 to 50 amino acids, for example 50 to 75 aminoacids, such as 75 to 100 amino acids, for example 100 to 125 aminoacids, such as 125 to 150 amino acids, for example 150 to 175 aminoacids, such as 175 to 200 amino acids, for example 200 to 225 aminoacids, such as 225 to 250 amino acids, for example 250 to 275 aminoacids, such as 275 to 300 amino acids, for example 300 to 325 aminoacids, such as 325 to 350 amino acids, for example 350 to 375 aminoacids, such as 375 to 400 amino acids, for example 400 to 425 aminoacids, such as 425 to 450 amino acids, for example 450 to 475 aminoacids, such as 475 to 500 amino acids, for example 500 to 525 aminoacids, such as 525 to 550 amino acids, for example 550 to 575 aminoacids, such as 575 to 600 amino acids, for example 600 to 625 aminoacids, such as 625 to 640 amino acids.

In one particular embodiment, the fragment or variant of Hsp70 comprisesall or part of the ATPase domain of Hsp70. It follows that the fragmentor variant of Hsp70 according to the present invention in one embodimentcomprises all or part of amino acids number 30 to 382.

In another particular embodiment, the fragment or variant of Hsp70comprises tryptophan at amino acid position 90 of the Hsp70 ATPasedomain.

A fragment of Hsp70 may be a truncated version of the wild type protein,meaning that it is a shorter version. A fragment may be truncated byshortening of the protein from either the amino-terminal or thecarboxy-terminal ends of the protein, or it may be truncated by deletionof one or more internal regions of any size of the protein.

A fragment or variant of Hsp70 may in one embodiment have 100% homologyto the wild-type protein. In another embodiment, the fragment or variantof Hsp70 may also be a variant of Hsp70 which has less than 100%homology to the wild-type protein, such as between 99.9 to 95% homology,for example 95 to 90% homology, such as 90 to 85% homology, for example85 to 80% homology, such as 80 to 75% homology, for example 75 to 60%homology to the wild-type protein.

It is to be understood that any fragment or variant of Hsp70 whichretains its ability to modulate lysosomal enzyme activity is encompassedby the present invention.

It is to be understood that any fragment or variant of Hsp70 whichretains its ability to interact with BMP is encompassed by the presentinvention.

It is appreciated that the exact quantitative effect of the functionalfragment or variant may be different from the effect of the full-lengthmolecule. In some instances, the functional fragment or variant mayindeed be more effective than the full-length molecule. Furthermore, theuse of fragments instead of full-length molecules may be advantageous inview of the smaller size of the fragments.

In one embodiment, a functional fragment or variant of Hsp70 may be avariant of Hsp70 in which one or more amino acids has been substituted.Said substitution(s) may be an equivalent or conservativesubstitution(s), or a non-equivalent or non-conservativesubstitution(s).

In one embodiment, between 0.1 to 1% of the amino acid residues of wildtype Hsp70 has been substituted, such as between 1 to 2% amino acidresidues, for example between 2 to 3% amino acid residues, such asbetween 3 to 4% amino acid residues, for example between 4 to 5% aminoacid residues, such as between 5 to 10% amino acid residues, for examplebetween 10 to 15% amino acid residues, such as between 15 to 20% aminoacid residues, for example between 20 to 30% amino acid residues, suchas between 30 to 40% amino acid residues, for example between 40 to 50%amino acid residues, such as between 50 to 60% amino acid residues, forexample between 60 to 70% amino acid residues, such as between 70 to 80%amino acid residues, for example between 80 to 90% amino acid residues,such as between 90 to 100% amino acid residues.

In one embodiment, between 1 to 5 of the amino acid residues of wildtype Hsp70 has been substituted, such as between 5 to 10 amino acidresidues, for example between 10 to 15 amino acid residues, such asbetween 15 to 20 amino acid residues, for example between 20 to 30 aminoacid residues, such as between 30 to 40 amino acid residues, for examplebetween 40 to 50 amino acid residues, such as between 50 to 75 aminoacid residues, for example between 75 to 100 amino acid residues, suchas between 100 to 150 amino acid residues, for example between 150 to200 amino acid residues, such as between 200 to 300 amino acid residues,for example between 300 to 400 amino acid residues, such as between 400to 500 amino acid residues.

In one embodiment, the functional fragment or variant of Hsp70 is afusion protein. In one embodiment, said functional fragment or variantof Hsp70 is fused to a tag.

Advantages of Using Hsp70, or a Functional Fragment or Variant Thereof

As discussed herein above, there are no cures for the lysosomal storagediseases and treatment is mostly symptomatic, with the exception of thedevelopment of enzyme replacement therapies (ERT) for Gaucher diseaseand Fabry disease. As mentioned, ERT is a very expensive form of therapythat is effective for one specific disease only.

To the knowledge of the inventors, to date no successful attempt hasbeen made to provide ERT for the remaining lysosomal storage diseasesassociated with lipid accumulation, thus a major unmet need for aneffective and specific treatment of these LSDs remains today.

Administration of Hsp70, or a functional fragment or variant thereof, toan individual in need thereof has a number of advantages compared toconventional treatment modalities for the lysosomal storage disorders.

First, producing a recombinant protein, such as rHsp70 or a functionalfragment or variant thereof, is with modern technology a simple andstraight-forward way of producing sufficient amounts of rHsp70, or afunctional fragment or variant thereof. Conventional techniques forproducing recombinant enzymes are well known to the skilled person.

Further, producing a recombinant protein, such as rHsp70 a functionalfragment or variant thereof, is a cheap method for producing sufficientamounts of rHsp70, or a functional fragment or variant thereof. Comparedto the production of enzymes for ERT, the cost is drastically reduced.

Also, the use of Hsp70, or a functional fragment or variant thereof canbe used for treatment of more than one specific lysosomal storagedisorder. This applies also to the Hsp70 inducers and co-inducers of thepresent invention. Indeed, the bioactive agent capable of increasing theintracellular concentration and/or activity of Hsp70 may be used fortreatment of any lysosomal storage disease which may be reverted bymodulating the enzymatic activity of the involved defective enzyme,wherein said enzyme interacts with BMP.

Finally, as Hsp70 is an endogenously occurring molecule, i.e. a moleculethat originate from within an organism, tissue, or cell, it is to beexpected that no or a very limited immune response is triggered byadministering Hsp70, or a functional fragment or variant thereof.

This is a major advantage as it facilitates treatment and reducespotential side effects when administered to an individual.

Ectopic Expression of Hp70

In one embodiment, Hsp70, or a functional fragment or variant thereof,may be expressed from a vector. The invention thus in one embodimentrelates to a vector encoding Hsp70, or a functional fragment or variantthereof.

In one embodiment of the present invention, Hsp70, or a functionalfragment or variant thereof, may be administered to an individual inneed thereof in the form of a vector.

The vector used for expressing Hsp70, or a functional fragment orvariant thereof, may be selected from the group consisting of: viralvectors (retroviral and adenoviral) or non-viral vectors (plasmid,cosmid, bacteriophage).

In one embodiment, said vector comprises one or more of a origin ofreplication, a marker for selection and one or more recognition sitesfor a restriction endonuclease. In another embodiment, said vector isoperably linked to regulatory sequences controlling the transcription ofsaid Hsp70, or a functional fragment or variant thereof, in a suitablehost cell.

The present invention in one embodiment relates to a method forproducing Hsp70, or a functional fragment or variant thereof, asdescribed herein; said method comprising the steps of providing a vectorencoding said Hsp70, or a functional fragment or variant thereof, andexpressing said vector either in vitro, or in vivo in a suitable hostorganism, thereby producing said Hsp70, or a functional fragment orvariant thereof.

The invention further relates to an isolated recombinant or transgenichost cell comprising a vector encoding Hsp70, or a functional fragmentor variant thereof, according to the present invention.

The invention also relates to a method for generating a recombinant ortransgenic host cell, said method comprising the steps of providing avector encoding Hsp70, or a functional fragment or variant thereof,introducing said vector into said recombinant or transgenic host celland optionally also expressing said vector in said recombinant ortransgenic host cell, thereby generating a recombinant or transgenichost cell producing said Hsp70, or a functional fragment or variantthereof.

In another embodiment the present invention relates to a transgenic,mammalian organism comprising the host cell described above.

In a further embodiment, the transgenic, mammalian organism comprisingthe recombinant or transgenic host cell according to the presentinvention is non-human.

The transgenic host cell may be selected from the group consisting of amammalian, plant, bacterial, yeast or fungal host cell.

To improve the delivery of the DNA into the cell, the DNA must beprotected from damage and its entry into the cell must be facilitated.Lipoplexes and polyplexes, have been created that have the ability toprotect the DNA from undesirable degradation during the transfectionprocess. Plasmid DNA can be covered with lipids in an organizedstructure like a micelle or a liposome. When the organized structure iscomplexed with DNA it is called a lipoplex.

There are three types of lipids that may be employed for formingliposomes; anionic (negatively charged), neutral, or cationic(positively charged). Complexes of polymers with DNA are calledpolyplexes. Most polyplexes consist of cationic polymers and theirproduction is regulated by ionic interactions.

In one embodiment, the vector comprising Hsp70, or a functional fragmentor variant thereof, may be used for gene therapy. Gene therapy is theinsertion of genes into an individual's cells and tissues to treat adisease, such as a hereditary disease in which a deleterious mutantallele is replaced with a functional one.

In another embodiment, Hsp70, or a functional fragment or variantthereof, may be administered as naked DNA. This is the simplest form ofnon-viral transfection. Delivery of naked DNA may be performed by use ofelectroporation, sonoporation, or the use of a “gene gun”, which shootsDNA coated gold particles into a cell using high pressure gas.

Bioactive Agent—Hsp70 Inducers and Co-Inducers

The present invention relates in one embodiment to the modulation ofenzymatic activity, wherein said enzyme interacts with BMP, by the useof Hsp70 inducers or co-inducers.

A Hsp70 inducer is a compound that can by itself amplify Hsp70 geneexpression and protein expression without a concomitant stress.

A Hsp70 co-inducer is a compound that cannot amplify Hsp70 geneexpression and protein expression without a concomitant (mild) stress,but the stress-induced increase in Hsp70 levels is further elevated orenhanced by their presence.

It is an aspect of the present invention to provide an Hsp70 inducer orco-inducer for use as a medicament.

It is a further aspect of the present invention to provide an Hsp70inducer or co-inducer for use in treating lysosomal storage disorders.

It is a still further aspect of the present invention to provide the useof an Hsp70 inducer or co-inducer, for the manufacture of a medicamentfor treating lysosomal storage disorders.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

In a particular embodiment, said lysosomal storage disorder isNiemann-Pick disease type A or B. In another particular embodiment, saidlysosomal storage disorder is Farber disease. In another particularembodiment, said lysosomal storage disorder is Krabbe disease. Inanother particular embodiment, said lysosomal storage disorder isMetachromatic leukodystrophy. In another particular embodiment, saidlysosomal storage disorder is Sialidosis. In another particularembodiment, said lysosomal storage disorder is Fabry disease. In yetanother particular embodiment, said lysosomal storage disorder isGaucher disease. In yet another particular embodiment, said lysosomalstorage disorder is saposin-deficiency.

In one embodiment, the bioactive agent according to the presentinvention is an Hsp70 inducer or co-inducer. In a particular embodiment,the bioactive agent according to the present invention is an Hsp70inducer. In another particular embodiment, the bioactive agent accordingto the present invention is an Hsp70 co-inducer.

Small-Molecule Drugs—Hydroxylamine Derivatives

In one embodiment, the bioactive agent according to the presentinvention is a Hsp70 co-inducer. In a further embodiment, said Hsp70co-inducer is a small-molecule drug.

In a particular embodiment, the Hsp70 co-inducer according to thepresent invention is a hydroxylamine derivative. Said hydroxylaminederivative may in a further embodiment selected from the group ofBimoclomol (BRLP-42), Arimoclomol (BRX-220), BRX-345 and BGP-15.

In a particular embodiment, said hydroxylamine derivative is Arimoclomol(BRX-220).

Bimoclomol ([2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidoyl-chloride maleate) is a non-toxiccompound that was originally developed for treatment of diabeticcomplications such as neuropathies. Bimoclomol has been shown to improvecell survival under experimental stress conditions partly by increasingintracellular heat shock proteins (HSPs), including Hsp70, via anactivation of HSF-1. It has been shown that bimoclomol possess thecapability of Hsp70 co-induction in the absence of unfolded proteins,and that bimoclomol interacts with and increases the fluidity ofnegatively charged membrane lipids. BRX-345 is a structural analog ofbimoclomol with a somewhat lesser ability to induce HSPs.

Arimoclomol (BRX-220) is an analog of bimoclomol, which also interactswith and amplifies the heat shock response. Arimoclomol is currently inclinical trials for the treatment of ALS (amyotrophic lateralsclerosis); a progressive neurodegenerative disorder. Arimoclomol isowned by CytRx Corporation.

It is thus an aspect of the present invention to provide a hydroxylaminederivative Hsp70 co-inducer for use in treating lysosomal storagedisorders.

It is a still further aspect of the present invention to provide the useof a hydroxylamine derivative Hsp70 co-inducer for the manufacture of amedicament for treating lysosomal storage disorders.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

Membrane Fluidiers

In one embodiment, the bioactive agent according to the presentinvention is a Hsp70 inducer. In a further embodiment, said Hsp70inducer is a membrane fluidizer.

Treatment with a membrane fluidizer may also be termed lipid therapy.

In a particular embodiment, the Hsp70 inducer according to the presentinvention is a membrane fluidizer selected from the group of benzylalcohol, heptanol, AL721, Docosahexaenoic acid, aliphatic alcohols,oleyl alcohol, dimethylaminoethanol, A2C, farnesol and anaesthetics suchas lidocaine, ropivacaine, bupivacaine and mepivacaine, as well asothers known to the skilled person.

Besides the denaturation of a proportion of cellular proteins duringheat (proteotoxicity), a change in the fluidity of membranes is alsoproposed as being a cellular thermosensor that initiates the heat shockresponse and induces HSPs. Indeed, chemically induced membraneperturbations—analogous with heat induced plasma membranefluidization—are capable of activating HSP, without causing proteindenaturation.

Membrane fluidity refers to the viscosity of the lipid bilayer of a cellmembrane. The membrane phospholipids incorporate fatty acids of varyinglength and saturation.

The membrane fluidizers act by intercalating between membrane lipidsthus inducing a disordering effect by weakening of van der Vaalsinteractions between the lipid acyl chains.

It is thus an aspect of the present invention to provide a membranefluidizer selected from the group of benzyl alcohol, heptanol, AL721,Docosahexaenoic acid, aliphatic alcohols, oleyl alcohol,dimethylaminoethanol, A₂C, farnesol and anaesthetics such as lidocaine,ropivacaine, bupivacaine and mepivacaine, as well as others known to theskilled person, for use in treating lysosomal storage disorders.

It is a still further aspect of the present invention to provide the useof a membrane fluidizer selected from the group of benzyl alcohol,heptanol, AL721, Docosahexaenoic acid, aliphatic alcohols, oleylalcohol, dimethylaminoethanol, A2C, farnesol and anaesthetics such aslidocaine, ropivacaine, bupivacaine and mepivacaine, as well as othersknown to the skilled person, for the manufacture of a medicament fortreating lysosomal storage disorders.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

Other Means for Inducing Hsp70

Any means for inducing Hsp70 expression is envisioned to be encompassedby the present invention, some of which are outlined herein below.

Increasing the temperature of an individual is a potent inducer of HSPsincluding Hsp70, and as such sub-lethal heat therapy is an aspect of thepresent invention. In one embodiment, sub-lethal heat therapy comprisesincreasing the temperature of an individual to a core temperature ofabout 38° C., such as about 39° C., for example about 40° C., such asabout 41° C., for example about 42° C., such as about 43° C.

It is thus an aspect of the present invention to provide sub-lethal heattherapy for use in treating lysosomal storage disorders.

Psychological stress such as predatory fear and electric shock can evokea stress induced eHsp70 release, a process which is suggested to bedependent on cathecholamine signaling. Further, adrenaline andnoradrenalin can evoke Hsp70 release.

The following compounds have been shown to induce (or co-induce) HSPs,including Hsp70: the membrane-interactive compound alkyllysophospholipidEdelfosine (ET-18-OCH3 or1-octadecyl-2-methyl-rac-glycero-3-phosphocholine); anti-inflammatorydrugs including cyclooxygenase 1/2 inhibitors such as celecoxib androfecoxib, as well as NSAIDs sub as acetyl-salicylic acid, sodiumsalicylate and indomethacin; prodstaglandins PGA1, PGj2 and2-cyclopentene-1-one; peroxidase proliferator-activated receptor-gammaagonists; tubulin-interacting anticancer agents including vincristineand paclitaxel; the insulin sensitizer pioglitazone; anti-neoplasticagents such as carboplatin, doxorubicin, fludarabine, ifosfamide andcytarabine; the Hsp90 inhibitors geldanamycin, 17-AAG, 17-DMAG,radicicol, herbimycin-A and arachidonic acid; proteasome inhibitorsMG132 and lactacystin; serine protease inhibitors DCIC, TLCK and TPCK;the anti-ulcer drugs geranylgeranylacetone (GGA), rebamipide,carbenoxolone and polaprezinc (zinc L-camosine); heavy metals (zinc andtin); the anti-inflammatory drug dexamethasone; cocaine; nicotine;alcohol; alpha-adrenergic agonists; cyclopentenone prostanoids; as wellas herbal medicines paeoniflorin, glycyrrhizin, celastrol,dihydrocelastrol, dihydrocelastrol diacetate and curcumin.

It is thus an aspect of the present invention to provide a compoundselected from the group of Edelfosine (ET-18-OCH3 or1-octadecyl-2-methyl-rac-glycero-3-phosphocholine), celecoxib,rofecoxib, acetyl-salicylic acid, sodium salicylate, indomethacin, PGA1,PGj2 2-cyclopentene-1-one, peroxidase proliferator-activatedreceptor-gamma agonists, vincristine, paclitaxel, pioglitazone,carboplatin, doxorubicin, fludarabine, ifosfamide cytarabine,geldanamycin, 17-AAG, 17-DMAG, radicicol, herbimycin-A, arachidonicacid, MG132, lactacystin, DCIC, TLCK, TPCK, geranylgeranylacetone (GGA),rebamipide, carbenoxolone, polaprezinc (zinc L-carnosine),dexamethasone, cocaine, nicotine, alcohol, alpha-adrenergic agonists,cyclopentenone prostanoids, paeoniflorin, glycyrrhizin, celastrol,dihydrocelastrol, dihydrocelastrol diacetate and curcumin, as well asother HSP inducers known to the skilled person, for use in treatinglysosomal storage disorders.

Pharmaceutical Composition According to the Present Invention

The present invention relates to the modulation of enzymatic activity,wherein said enzyme interacts with BMP, by use of a bioactive agentcapable of increasing the concentration and/or activity of Hsp70,thereby benefiting patients suffering from lysosomal storage diseases.

Whilst it is possible for the bioactive agents of the present inventionto be administered as the raw chemical, it is preferred to present themin the form of a pharmaceutical formulation. Accordingly, the presentinvention further provides a pharmaceutical composition, for medicinalapplication, which comprises a bioactive agent of the present inventionor pharmaceutically acceptable salts thereof, as herein defined, and apharmaceutically acceptable carrier therefore.

It is an aspect of the present invention to provide a composition, suchas a pharmaceutical composition, comprising a bioactive agent identifiedherein that may be administered to an individual in need thereof.

In one embodiment, the invention relates to a composition comprising abioactive agent according to the present invention. The composition asdisclosed herein may in one embodiment be formulated in combination witha physiologically acceptable carrier. The composition as disclosedherein may in one embodiment be formulated in combination with apharmaceutically acceptable carrier.

Pharmaceutical compositions containing a bioactive agent of the presentinvention may be prepared by conventional techniques, e.g. as describedin Remington: The Science and Practice of Pharmacy 1995, edited by E. W.Martin, Mack Publishing Company, 19th edition, Easton, Pa.

The bioactive agents of the present invention may be formulated forparenteral administration and may be presented in unit dose form inampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, carriers, diluents, or solvents including aqueous solutions ofmineral salts or other water-soluble molecules, propylene glycol,polyethylene glycol, vegetable oils, animal oils, synthetic oils,injectable organic esters, and may contain formulatory agents such aspreserving, wetting, emulsifying or suspending, stabilizing and/ordispersing agents, colorants, buffers, thickeners, solubilizing agentsand the like. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilization from solution for constitution before use with a suitablevehicle, e.g., sterile, pyrogen-free water.

Pharmaceutically acceptable salts of the bioactive agents, where theycan be prepared, are also intended to be covered by this invention, asare specific hydrate forms of a salt. These salts will be ones which areacceptable in their application to a pharmaceutical use. By that it ismeant that the salt will retain the biological activity of the parentcompound and the salt will not have untoward or deleterious effects inits application and use in treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. Ifthe parent compound is a base it is treated with an excess of an organicor inorganic acid in a suitable solvent. If the parent compound is anacid, it is treated with an inorganic or organic base in a suitablesolvent.

Any suitable formulation of the bioactive agent according to the presentinvention may be employed, known to the skilled person.

In one embodiment, the Hsp70, or a functional fragment or variantthereof, is formulated in a biodegradable microsphere, such as aliposome.

Administration

Any suitable route of administration may be employed for providing amammal, preferably a human, with an effective amount of a bioactiveagent according to the present invention, wherein said bioactive agentmay be Hsp70, or a functional fragment or variant thereof.

Administering bioactive agents or pharmaceutical compositions to anindividual in need thereof may occur via three major routes ofdelivery: 1) Topical (applied to body surfaces such as skin or mucousmembranes), 2) Enteral (via the gastrointestinal or digestive tract) and3) Parenteral (routes other than the gastrointestinal or digestivetract).

Topical administration includes epicutaneous (application onto theskin), inhalational, enema, eye drops (onto the conjunctiva), ear drops,intranasal route, and vaginal administration.

Enteral administration is any form of administration that involves anypart of the gastrointestinal tract and includes oral administration (bymouth e.g. tablets, capsules or drops), intrarectal (e.g. suppository orenema) administration besides by gastric or duodenal feeding tube.

Parenteral delivery, such as by injection or infusion, are effective todeliver the bioactive agent to a target site or to introduce the druginto the bloodstream, and includes intravenous (into a vein),intra-arterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), subcutaneous (under the skin),intraosseous (into the bone marrow), intradermal, (into the skinitself), intrathecal or intraspinal (into the spinal canal),intraperitoneal, (into the peritoneum), transdermal (diffusion throughthe intact skin), transmucosal (diffusion through a mucous membrane,e.g. insufflation (snorting), sublingual, buccal and vaginalsuppositories), inhalational, epidural (into the epidural space) andintravitreal (into the eye).

Sublingual administration (under the tongue) is also a form ofparenteral administration, whereby bioactive agents diffuse into thebloodstream through the mucosal tissue under the tongue. The bioactiveagent of the present invention may be administered by any parenteralroute of delivery and preferably any of the above.

Parenteral delivery has the advantage of avoiding degradation in thegastrointestinal tract, as associated with enteral delivery.

Parenteral delivery has the further advantage of of abolishing firstpass metabolism, as associated with enteral delivery, because it allowscompounds to be absorbed directly into the systemic circulation.

First-pass metabolism is a phenomenon of drug metabolism whereby theconcentration of a drug is greatly reduced before it reaches thesystemic circulation. It is the fraction of lost drug during the processof absorption which is generally related to the liver and gut wall.

After a drug is swallowed, it is absorbed by the digestive system andenters the hepatic portal system. It is carried through the portal veininto the liver before it reaches the rest of the body. The livermetabolizes many drugs, sometimes to such an extent that only a smallamount of active drug emerges from the liver to the rest of thecirculatory system. This first pass through the liver thus greatlyreduces the bioavailability of the drug.

The four primary systems that affect the first pass effect of a drug arethe enzymes of the gastrointestinal lumen, gut wall enzymes, bacterialenzymes, and hepatic enzymes.

Appropriate dosage forms for such administration may be prepared byconventional techniques. Appropriate dosage forms for administration byinhalation, such as an aerosol formulation or a metered dose inhaler,may be prepared by conventional techniques.

In one embodiment, a particular mode of administration of a bioactiveagent according to the present invention is by parenteraladministration.

In one embodiment, a particular mode of parenteral administration of abioactive agent of the present invention is by intravenous,subcutaneous, intramuscular, intraarterial, subcutaneous orintraperitoneal injection.

In one embodiment, a particular mode of parenteral administration of abioactive agent of the present invention is by inhalation.

In one embodiment, a particular mode of parenteral administration of abioactive agent of the present invention is by intravenous infusion.

Intravenous infusion according to the present invention may in oneembodiment occur over a time period of from 10 minutes to 20 minutes,such as 20 to 30 minutes, for example 30 to 40 minutes, such as 40 to 50minutes, for example 50 to 60 minutes, such as 60 to 90 minutes, forexample 90 to 120 minutes, such as 2 hours to 3 hours, for example 3 to4 hours, such as 4 to 5 hours, for example 5 to 6 hours, such as 6 to 7hours, for example 7 to 8 hours.

In a particular embodiment, the mode of parenteral administration of abioactive agent of the present invention is by transmucosal delivery.Said transmucosal delivery is in one embodiment sublingual delivery, inanother embodiment said transmucosal delivery is buccal delivery, and inyet another embodiment said transmucosal delivery is insufflation orintranasal delivery.

Dosage forms include tablets, troches, dispersions, suspensions,solutions, capsules, creams, ointments, emulsions, gels, lotions,pastes, aerosols, or other forms known in the art.

The effective dosage of active ingredient employed may vary depending onthe particular composition employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Such dosage may be ascertained readily by a person skilled in the art.

In one embodiment, the bioactive agent of the present invention isadministered at a daily dosage of from about 1 microgram to about 100milligram per kilogram of animal body weight, given as a single dailydose or in divided doses, or in sustained release form. The dosageregimen may be adjusted within this range or even outside of this rangeto provide the optimal therapeutic response.

In one embodiment, the bioactive agent of the present invention isadministered at a dosage of from about 1 μg to about 10 μg per kg bodyweight, such as from about 10 μg to about 50 μg per kg body weight, forexample from about 50 μg to about 100 μg per kg body weight, such asfrom about 100 μg to about 250 μg per kg body weight, for example fromabout 250 μg to about 500 μg per kg body weight, such as from about 500μg to about 750 μg per kg body weight, for example from about 750 μg toabout 1000 μg per kg body weight, such as from about 1 mg to about 10 mgper kg body weight, for example from about 10 mg to about 50 mg per kgbody weight, such as from about 50 mg to about 100 mg per kg bodyweight.

Said dosage may be administered in certain time intervals, and may beexpressed as mg per kg body weight per time unit. Said time unit may inone embodiment be per minute, such as per hour, for example per day,such as per week.

Combination Treatment

It is an aspect of the present invention to provide a bioactive agentcapable of increasing the intracellular concentration and/or activity ofHsp70 for use in treatment of lysosomal storage disorders, incombination with other treatment modalities.

The present invention in one aspect relates to a method of treatment ofa lysosomal storage disease comprising administration of the bioactiveagent according to any the present invention in combination with atleast one other treatment modality.

Thus, in one embodiment, the bioactive agent according to the presentinvention is administered to an individual in need thereof incombination with at least one other treatment modality, such asconventional or known treatment modalities for LSDs.

It is understood, that the bioactive agent according to the presentinvention is Hsp70 or a functional fragment or variant thereof, or anHsp70 inducer or co-inducer.

Administering more than one treatment modality in combination may occureither simultaneously, or sequentially. Simultaneous administration maybe two compounds comprised in the same composition or comprised inseparate compositions, or may be one composition and one other treatmentmodality performed essentially at the same time.

Sequential administration means that the more than one treatmentmodalities are administered at different time points, such asadministering one treatment modality first, and administering the secondtreatment modality subsequently. The time frame for administering morethan one treatment modality sequentially may be determined by a skilledperson in the art for achieving the optimal effect, and may in oneembodiment be between 30 minutes to 72 hours.

The treatment modalities in the form of chemical compounds may beadministered together or separately, each at its most effective dosage.Administering more than one compound may have a synergistic effect, thuseffectively reducing the required dosage of each drug.

In one embodiment, the bioactive agent according to the presentinvention is administered to an individual in need thereof incombination with enzyme replacement therapy (ERT). Said ERT may in oneembodiment be selected from the group consisting of Cerezyme®(imiglucerase for injection), Miglustat, Fabrazyme® (agalsidase beta),and Replagal (Agalsidase alpha).

In one embodiment, the bioactive agent according to the presentinvention is administered to an individual with Gaucher disease incombination with Cerezyme® (imiglucerase for injection) or Miglustat.

In another embodiment, the bioactive agent according to the presentinvention is administered to an individual with Fabry disease incombination with Fabrazyme® (agalsidase beta) or Replagal (Agalsidasealpha).

In another embodiment, the bioactive agent according to the presentinvention is administered to an individual in need thereof incombination with pain relievers.

In yet another embodiment, the bioactive agent according to the presentinvention is administered to an individual in need thereof incombination with corticosteroids.

The bioactive agent according to the present invention may in oneembodiment be administered to an individual in need thereof incombination with a transplantation, such as bone marrow transplantation,cord blood transplantation or stem cell transplantation.

The bioactive agent according to the present invention may in anotherembodiment be administered to an individual in need thereof incombination with substrate reduction therapy.

In another embodiment, the bioactive agent according to the presentinvention is administered to an individual in need thereof incombination with symptomatic and supportive therapy, such as physicaltherapy.

Hsp70 Increases the Uptake of Compounds

The present inventors have further shown that Hsp70 increases theendocytic uptake of other molecules (FIG. 16). This increased uptake mayoccur independently on Hsp70 due to a passive mechanism which allows acompound to be more readily taken up by the cell in the presence ofHsp70, or it may be occur dependently on Hsp70 due to a directassociation with Hsp70.

The ability of Hsp70 to increase the cellular uptake of compounds is anadvantage in that it allows for Hsp70, or a functional fragment orvariant thereof, administered to cells to be readily taken up by thecell.

Further, the ability of Hsp70 to increase the cellular uptake ofcompounds is an advantage in combination treatment regimens, as thepresence of Hsp70 may increase the uptake of both Hsp70 and the compoundgiven in combination with Hsp70.

In respect to combination therapy wherein one compound is an enzyme forERT, and the other is Hsp70, or a functional fragment or variantthereof, this may help effectively reduce the amount of enzyme for ERTneeded to achieve an effective intracellular dosis. This is relevant asERT is very expensive.

In the situation in which the bioactive agent according to the presentinvention comprises a combination of Hsp70, or a functional fragment orvariant thereof, and an Hsp70 inducer or co-inducer, the presence ofHsp70 may therefore increase the uptake of said Hsp70 inducer orco-inducer.

Method for Modulating the Enzymatic Activity of an Enzyme

The present invention relates in one aspect to the modulation ofenzymatic activity. Said enzyme may be an enzyme involved in thecatabolism of lysosomal substances. And said modulation may derive froman interaction between Hsp70 and BMP.

The present inventors have thus described an interaction between Hsp70and BMP, wherein Hsp70 interacts with or binds to BMP with a certainaffinity. By a molecule having an “affinity” for molecule X is meantherein that a molecule with affinity for molecule X will bind tomolecule X in a certain detectable amount under certain conditions butwill not (optionally detectably) bind other, different molecules (forwhich it does not have affinity for) to the same extent under identicalconditions. One measure to describe a molecule's affinity to anothermolecule is a dissociation constant, Kd. The smaller the Kd, thestronger the affinity. Dissociation constants can be determined usingmethods well-known in the art, such as surface plasmon resonanceanalysis. Herein, it is preferred that a molecule with “affinity” foranother molecule X has a Kd for said molecule X that is less than 100mM, such as less than 10 mM, for example less than 5 mM, such as lessthan 1 mM, for example less than 0.1 mM, such as less than 0.01 mM, forexample less than 1 μM, such as less than 100 nM, for example less than10 nM, such as less than 1 nM, for example less than 100 μM, such asless than 10 μM, for example less than 1 μM. Furthermore, it is hereinpreferred that a molecule that “does not have an affinity” to molecule Xhas a dissociation constant, Kd with respect to binding molecule X thatis at least 10 fold larger, such as at least 20 fold larger, for exampleat least 30 fold larger, such as at least 40 fold larger, for example atleast 50 fold larger, such as at least 60 fold larger, for example atleast 70 fold larger, such as at least 80 fold larger, for example atleast 90 fold larger, such as at least 100 fold larger, than the Kd ofthe binding (to molecule X) of a molecule that does have affinity tomolecule X. Most preferably, there is at least a ten-fold difference inKd between those molecules considered to have an affinity and thosedeemed not to have an affinity to a molecule X.

It is an aspect of the present invention to provide a method formodulating the enzymatic activity of an enzyme, wherein said enzymeinteracts with BMP (bis(monoacylglycero)phosphate), said methodcomprising the steps of

-   -   i) administering a bioactive agent capable of increasing the        intracellular concentration and/or activity of Hsp70, and    -   ii) allowing interaction between BMP and Hsp70, and    -   iii) modulating the enzymatic activity of an enzyme interacting        with BMP.

Said interaction may in one embodiment be direct, or said interactionmay in another embodiment be indirect.

In one embodiment, the present invention relates to a method formodulating the enzymatic activity of an enzyme, wherein said enzymeinteracts with BMP (bis(monoacylglycero)phosphate), said methodcomprising the steps of

-   -   i) administering the bioactive agent according to the present        invention,    -   ii) allowing interaction between BMP and Hsp70, and    -   iii) modulating the enzymatic activity of an enzyme interacting        with BMP.

In one embodiment, said Hsp70 forms a covalent or non-covalent complexwith BMP.

In one embodiment, said BMP interacts with a saposin. In a furtherembodiment, said saposin may be selected from the group consisting ofsaposin A, saposin B, saposin C, and saposin D.

In a further embodiment, said enzyme is selected from the groupconsisting of sphingomyelinase, acidic sphingomyelinase (aSMase), acidceremidase, beta-galactosylceremidase, alpha-galactosidase,beta-galactosidase, glucosylceremidase, sialidase and aryl sulfatase.

In one particular embodiment, the modulation of the enzymatic activityis an increase in the enzymatic activity.

In one embodiment, said increase in the enzymatic activity is anincrease in the range of 1 to 5%, such as in the range of 5 to 10%, forexample in the range of 10 to 15%, such as in the range of 15 to 20%,for example in the range of 20 to 25%, such as in the range of 25 to30%, for example in the range of 30 to 35%, such as in the range of 35to 40%, for example in the range of 40 to 45%, such as in the range of45 to 50%/c, for example in the range of 50 to 60%, such as in the rangeof 60 to 70%, for example in the range of 70 to 80%, such as in therange of 80 to 90%, for example in the range of 90 to 100%, such as inthe range of 100 to 120%, for example in the range of 120 to 140%, suchas in the range of 140 to 160%, for example in the range of 160 to 180%,such as in the range of 180 to 200%, for example in the range of 200 to250%, such as in the range of 250 to 300%, for example in the range of300 to 400%, such as in the range of 400 to 500%, for example in therange of 500 to 750%, such as in the range of 750 to 1000%, for examplein the range of 1000 to 1500%, such as in the range of 1500 to 2000%,for example in the range of 2000 to 5000%.

The present invention in another aspect relates to a method foridentifying binding partners for the Hsp70-BMP complex, said methodcomprising the steps of extracting said Hsp70-BMP complex and isolatingsaid binding partners. In one embodiment, said binding artner is anagonist. In another embodiment, said binding partner is an antagonist.

The present invention in another aspect relates to a Hsp70-BMP complex,and its use for a medicament, such as for the treatment of a lysosomalstorage disease.

In one embodiment, the present invention relates to an antibody thatspecifically recognizes the Hsp70-BMP complex.

Method of Treatment

The present invention relates in one aspect to a method for treating anindividual in need thereof.

It is thus an aspect of the present invention to provide a method fortreatment of a lysosomal storage disease comprising administration ofthe bioactive agent according to the present invention to an individualin need thereof.

It follows, that in one embodiment said treatment may be prophylactic,curative or ameliorating. In one particular embodiment, said treatmentis prophylactic. In another embodiment, said treatment is curative. In afurther embodiment, said treatment is ameliorating.

The bioactive agent used according to the present invention may in oneembodiment be formulated as a pharmaceutical composition.

In one embodiment, said lysosomal storage disorder is selected from thegroup consisting of Niemann-Pick disease, Farber disease, Krabbedisease, Fabry disease, Gaucher disease, Metachromatic leukodystrophy,Sialidosis and saposin-deficiency.

In a particular embodiment, said lysosomal storage disorder isNiemann-Pick disease type A or B. In another particular embodiment, saidlysosomal storage disorder is Farber disease. In another particularembodiment, said lysosomal storage disorder is Krabbe disease. Inanother particular embodiment, said lysosomal storage disorder isMetachromatic leukodystrophy. In another particular embodiment, saidlysosomal storage disorder is Sialidosis. In another particularembodiment, said lysosomal storage disorder is Fabry disease. In yetanother particular embodiment, said lysosomal storage disorder isGaucher disease. In yet another particular embodiment, said lysosomalstorage disorder is saposin-deficiency.

In one embodiment, said lysosomal disease is characterized by anincreased intracellular accumulation of a sphingolipid.

In one embodiment, said treatment reduces the intracellular accumulationof substances in an individual in need thereof. Said substance may be asubstance which is normally degraded in the lysosomes. In oneembodiment, said substance is a shingolipid.

In one embodiment, the treatment according to the present inventionreduces the intracellular accumulation of a lysosomally degradablesubstance such as a sphingolipid to less than 100% of the accumulatedamount, such than less than 90% of the accumulated amount, for exampleless than 80% of the accumulated amount, such than less than 70% of theaccumulated amount, for example less than 60% of the accumulated amount,such than less than 50% of the accumulated amount, for example less than40% of the accumulated amount, such than less than 30% of theaccumulated amount, for example less than 20% of the accumulated amount,such than less than 10% of the accumulated amount, for example less than5% of the accumulated amount.

In one embodiment, the treatment according to the present inventionreduces the intracellular accumulation of a sphingolipid by at least 5%,such as at least 10%, for example at least 15%, such as at least 20%,for example at least 25%, such as at least 30%, for example at least35%, such as at least 40%, for example at least 45%, such as at least50%, for example at least 55%, such as at least 60%, for example atleast 65%, such as at least 70%, for example at least 75%, such as atleast 80%, for example at least 85%, such as at least 90%, for exampleat least 95%, such as at least 1⁰⁰%.

In one embodiment, said accumulated sphingolipid is selected from thegroup consisting of sphingomyelin, ceramide, galactosylceramide,globotriaosylceramide, glycosylceramide, GM3 and sulfatide.

The rate of reducing the intracellular concentration of a lysosomalydegradable substance such as a sphingolipid may depend on factors suchas administration form, dosage regimens and the like.

In one embodiment, said treatment prolongs the life expectancy of saidindividual in need thereof.

It follows, that the life expectancy may in one embodiment be increasedby between 6 months to 1 year, such as from 1 year to 2 years, forexample from 2 to 3 years, such as from 3 to 4 years, for example from 4to 5 years, such as from 5 to 6 years, for example from 6 to 7 years,such as from 7 to 8 years, for example from 8 to 9 years, such as from 9to 10 years, for example from 10 to 12 years, such as from 12 to 14years, for example from 14 to 16 years, such as from 16 to 18 years, forexample from 18 to 20 years, such as from 20 to 25 years, for examplefrom 25 to 30 years, such as from 30 to 40 years, for example from 40 to50 years, such as from 50 to 60 years, for example from 60 to 70 years,such as from 70 to 80 years, for example from 80 to 90 years, such asfrom 90 to 100 years.

In one embodiment life expectancy is increased by at least 6 months,such as at least 1 year, such as at least 2 years, for example 3 years,such as at least 4 years, for example 5 years, such as at least 6 years,for example 7 years, such as at least 8 years, for example 9 years, suchas at least 10 years, for example 12 years, such as at least 14 years,for example 16 years, such as at least 18 years, for example 20 years,such as at least 25 years, for example 30 years, such as at least 40years, for example 50 years, such as at least 60 years, for example 70years, such as at least 80 years, for example 90 years, such as at least100 years.

It is also an aspect of the present invention to provide a method forprolonging life expectancy in a patient with a lysosomal storagedisease, wherein said method comprises administration of the bioactiveagent according to the present invention to an individual in needthereof.

In one embodiment, the present invention relates to a method forprolonging life expectancy in a patient with a lysosomal storagedisease, wherein said method comprises administration of the bioactiveagent according to the present invention to an individual in needthereof, wherein said life expectancy is increased by between 6 monthsto 1 year, such as from 1 year to 2 years, for example from 2 to 3years, such as from 3 to 4 years, for example from 4 to 5 years, such asfrom 5 to 6 years, for example from 6 to 7 years, such as from 7 to 8years, for example from 8 to 9 years, such as from 9 to 10 years, forexample from 10 to 12 years, such as from 12 to 14 years, for examplefrom 14 to 16 years, such as from 16 to 18 years, for example from 18 to20 years, such as from 20 to 25 years, for example from 25 to 30 years,such as from 30 to 40 years, for example from 40 to 50 years, such asfrom 50 to 60 years, for example from 60 to 70 years, such as from 70 to80 years, for example from 80 to 90 years, such as from 90 to 100 years.

In one embodiment, the present invention relates to a method forprolonging life expectancy in a patient with a lysosomal storagedisease, wherein said method comprises administration of the bioactiveagent according to the present invention to an individual in needthereof, wherein said life expectancy is increased by at least 6 months,such as at least 1 year, such as at least 2 years, for example 3 years,such as at least 4 years, for example 5 years, such as at least 6 years,for example 7 years, such as at least 8 years, for example 9 years, suchas at least 10 years, for example 12 years, such as at least 14 years,for example 16 years, such as at least 18 years, for example 20 years,such as at least 25 years, for example 30 years, such as at least 40years, for example 50 years, such as at least 60 years, for example 70years, such as at least 80 years, for example 90 years, such as at least100 years.

EXAMPLES Example 1: Interaction Between Hsp70 andBis(Monoacylglycero)Phosphate Stabilizes Lysosomes and Promotes CellSurvival

Abstract

Lysosomal membrane permeabilization is an evolutionarily conservedhallmark of stress-induced cell death. Here the inventors show that themajor stress-inducible heat shock protein 70 (Hsp70) enhances cellsurvival by stabilizing lysosomes through a pH-dependent high affinitybinding to an endo-lysosomal anionic phospholipidbis(monoacylglycero)phosphate (BMP; also referred to aslysobisphosphatidic acid). The positively charged ATPase domain of Hsp70is responsible for the binding but the substrate-binding domain is alsorequired for effective stabilization of lysosomes. Importantly, thecytoprotective effect can be obtained by endocytic delivery ofrecombinant Hsp70 and specifically reverted by extra cellularadministration of BMP antibodies or Hsp70 inhibitors. Thus, thisprotein-lipid interaction opens exciting possibilities for thedevelopment of cytoprotective and cytotoxic lysosome-specific therapiesfor the treatment of degenerative diseases and cancer, respectively.

Introduction

Lysosomes are highly dynamic cytosolic organelles that receive membranetraffic input from the biosynthetic (trans-Golgi network), endocytic,phagocytic and autophagic pathways. They contain over 50 acid hydrolasesthat can process all the major macromolecules of the cell to breakdownproducts available for metabolic reutilization. In addition to theircatabolic house keeping functions, lysosomal proteases, cathepsins, haverecently been identified as important effectors in evolutionarilyconserved cell death programs induced for example by death receptors oftumor necrosis factor receptor family, hypoxia, oxidative stress,osmotic stress, heat and anti-cancer drugs. Cathepsin-dependent celldeath is characterized by an early lysosomal membrane permeabilizationand the subsequent translocation of cathepsins into the cytosol, wherethey can initiate both caspase-dependent and -independent cell deathpathways. Thus, the lysosomal membrane integrity emerges as an importantregulator of cell survival during various stress conditions. Whereascytosolic cysteine protease inhibitors have been reported to conferprotection against cathepsin-induced cellular damage both in mammaliancells as well as in nematode (Caenorhabditis elegans, the mechanisms bywhich cells regulate lysosomal membrane stability have remained largelyobscure. Recent indirect evidence suggests, however, that the potentcytoprotective effect of the major stress-inducible Hsp70 is due tolysosomal membrane stabilization. The depletion of Hsp70 triggers anearly permeabilization of lysosomal membranes and cathepsin-mediatedcell death in cancer cells, and exogenous Hsp70 effectively inhibitslysosomal destabilization induced by various stresses. Furthermore, micedeficient for Hsp70 suffer from pancreatitis caused by the leakage oflysosomal proteases into the cytosol.

The molecular mechanism underlying the lysosome protective potential ofHsp70 has remained elusive, but clues to its mechanism of action may liein the stress- and cancer-associated translocation of a small portion ofHsp70 to the endo-lysososomal compartment. The major aim of this studywas to define whether the lysosomal localization, indeed, is crucial forthe cytoprotective effect of Hsp70. Remarkably, the data presentedherein demonstrate that Hsp70 binds with high affinity to alysosome-specific lipid BMP and that this protein-lipid interactionstabilizes lysosomes. Importantly this novel cytoprotective mechanismcan be exploited by extracellular administration of eithercytoprotective Hsp70 itself or compounds that interfere with Hsp70-BMPbinding or Hsp70 function specifically in the lysosomal compartment.

Results and Discussion

In order to test whether the lysosomal localization is crucial for thecytoprotective effect of Hsp70, the present inventors producedrecombinant Hsp70 (rHsp70) and took advantage of the endocytic machineryof cells to target rHsp70 into the lysosomal lumen. Immunocytochemicalanalysis of U-2-OS osteosarcoma cells incubated with Alexa Fluor488-labeled rHsp70 revealed a clear co-localization of the endocytosedrHsp70 with late endosomal and lysosomal marker proteins(lysosome-associated membrane proteins 1 and 2 and lysosomal integralmembrane protein-1 (LIMP-1)) and an endo-lysosome-specific lipid (BMP),whereas no co-localization was seen with markers for the endoplasmaticreticulum (endoplasmatic reticulum Ca²⁺-ATPase (SERCA)), golgi apparatus(golgin-97) or mitochondria (cytochrome c (cyt c)). The lysosomallocalization was also observed in living cells, where the endocytosedrHsp70 co-localized with Lysotracker® Red but not with Mitotracker® Red.In order to determine the amount of endocytosed Hsp70 the fluorescentsignal from the rHsp70*-loaded cells was quantified, which revealed thatan average of 70 ng rHsp70* is taken up pr. 1*10 cells. To determinewhether endocytosed rHsp70* was merely localized to the lumen or whetherit would have a direct attachment to the endo-lysosomal membranes, therHsp70*-loaded U-2-OS cells were sub-fractionated and the amount ofrHsp70* present in the light membrane fraction (LMF) measured (cellularorganelles including early and late endosomes and lysosomes). Freezefracturing of the organelles in the LMF via repeated freeze/thaw cyclesin liquid nitrogen, resulted in the total release of Cathepsin B intothe supernatant, whereas the lysosomal membrane protein LAMP-2 wasretained in the pelleted, fractured membrane fraction. Quantification ofthe endocytosed rHsp70* revealed that approx. ⅓ of the total rHsp70*remained in the pellet, strongly suggesting that it was bound to theendo-lysosomal membranes. In order to assess whether the endocytosedrHsp70 could stabilize the lysosomal membranes, cells were loaded withacridine orange, a metachromatic weak base that accumulates in theacidic compartment of the cells, i.e. late endosomes and lysosomes, andsensitizes them to photo-oxidation upon exposure to blue light (Brunk etal., 1997; Nylandsted et al., 2004). The photo-oxidation results in theloss of the lysosomal pH-gradient and leakage of acridine orange to thecytosol. This can be readily visualized and quantified as acridineorange exhibits red fluorescence when concentrated in the acidiccompartment of the cell and green fluorescence when at a lowerconcentration in the cytosol. Remarkably, the endocytosed rHsp70protected the lysosomes against blue light-induced photo-oxidation,whereas no protection was observed in cells loaded with recombinantHsc70 and Hsp70-2, which share 86% and 84% amino acid sequence homologywith Hsp70, respectively. Furthermore, a short interfering RNA (siRNA)specific for Hsp70 sensitized lysosomes of U-2-OS cells tophoto-oxidation. and this effect was fully reverted by endocytosedrHsp70 aptly demonstrating that the protective effect of endogenousHsp70 is mediated by the small fraction of the protein localized to thelysosomal lumen rather then the large pool residing in the cytosol. Theabove demonstrated effective endocytic uptake of Hsp70 and lysosomalstabilization may explain the recently reported surprisingneuroprotective effects of extra cellular Hsp70 administered to thesites of injury following a variety of treatments known to trigger thelysosomal cell death pathway, i.e. retinal light damage and sciaticnerve axotomy.

In order to test whether the protective effect of Hsp70 could be aconsequence of a direct association of Hsp70 with the lysosomalmembranes, the inventors investigated its interaction withpalmitoyl-oleoyl-phosphatidylcholine (POPC) large unilamellar vesicles(LUVs) containing a variety of membrane-associated anionic lipids, i.e.palmitoyl-oleoyl-phosphatidylserine (POPS; primarily in inner leaflet ofthe plasma membrane), cardiolipin (primarily mitochondrial) and BMP(primarily in late endosomes and lysosomes). Taking into account theincreasingly acidic milieu of the endo-lysosomal compartment uponmaturation to lysosomes, the protein-lipid interactions in neutral (pH7.4) and acidic (pH 6.0) conditions were compared. At pH 7.4, rHsp70caused a little relative change in the 90° light scattering in POPCliposomes indicating a very weak binding to the POPC bilayer. Asreported earlier for POPS, all negatively charged lipids enhanced thebinding of rHsp70 to the liposomes at neutral pH. This enhancement wasapproximately 4-fold irrespective of the negative lipid or the chargedensity on the liposome surface (POPS has a net charge of −1, andcardiolipin and BMP have a net charge of −2). Remarkably, lowering ofthe pH from 7.4 to 6.0 dramatically changed the lipid associationprofile of rHsp70. Whereas the binding to POPS was only slightlyincreased upon acidification, the binding to BMP was almost 20 timesstronger in the acidic pH as compared to the neutral pH. ThepH-dependent, high affinity binding of Hsp70 to BMP was confirmed in anindependent set of BIAcore experiments.

In order to test whether the pH-dependent high affinity interactionbetween Hsp70 and BMP observed in vitro was required for theHsp70-mediated stabilization of lysosomes in living cells, the inventorstargeted the cellular BMP by loading the endo-lysosomal compartment ofU-2-OS cells with BMP antibodies as demonstrated earlier (Kobayashi etal., 1998). Remarkably, BMP antibodies effectively inhibited the abilityof rHsp70 to confer protection against photooxidation-induced lysosomalleakage. Even more importantly, BMP antibodies significantly sensitizedU-2-OS osteosarcoma cells to cisplatin, which induces an early lysosomalmembrane permeabilization in U-2-OS cells as well as other cisplatinsensitive cell lines used in this study. Accordingly, also PC-3 andDU-145 prostate carcinoma cells were significantly sensitized tocisplatin-induced cell death upon treatment with anti-BMP antibodies.

Having confirmed that the lysosomal Hsp70-BMP interaction is essentialfor the cytoprotective effect of Hsp70, the inventors next investigatedwhich part of the Hsp70 protein is responsible for the lipid binding. Todetermine this, the fluorescence shift of the tryptophans (W90 and W580)upon docking of rHsp70 into BMP-containing liposomes at pH 6.0 wasmeasured. The inventors produced rHsp70 mutant proteins with deletionsof the two major functional domains of the protein, i.e. theamino-terminal ATPase domain (rHsp70-□ATP; deletion of amino acids119-426) and the carboxy-terminal peptide-binding domain (rHsp70-□PBD;deletion of amino acids 437-617). The loss of signal in relative peakfluorescence intensity for Hsp70-□ATP indicated that the ATPase domainis required for the high affinity binding of Hsp70 to the POPC/BMPbilayer. Next, the two tryptophans in Hsp70 were substituted withphenylalanines (W90F and W580F) in order to study which tryptophan isresponsible for the lipid binding and fluorescence shift. The reductionof the signal with rHsp70-W90F that lacks the tryptophan in the ATPasedomain (rHsp70-W90F) and the unchanged signal with rHsp70-W580F thatlacks the tryptophan in the peptide-binding domain indicated that thetryptophan in the position 90 docked into the lipid layer. As the methodused above only measured the relative shift in fluorescence upontryptophan embedding into the lipophilic environment, the inventors alsoanalyzed the lipid association of rHsp70 and its mutants in a morequantitative manner employing a BIAcore 2000 system with immobilizedBMP-containing LUVs on the surface of an L1 sensor chip at pH 4.5. BothrHsp70 and rHsp70-EPBD showed a strong interaction with BMP, whereas thebinding of rHsp70-ATP was markedly reduced confirming that Hsp70interacts with BMP mainly through its ATPase domain. Surprisingly, thetryptophan mutants showed a striking difference in their ability tointeract with BMP. Whereas the rHsp70-W580F mutant had essentially thesame interaction profile as rHsp70, the binding of rHsp70-W90F mutantwas dramatically decreased. Since rHsp70-W90F was properly folded asanalyzed by far- and near-UV circular dichroism and capable of foldingluciferace and hydrolyzing ATP, the W90F mutation specifically abolishedthe interaction between Hsp70 and BMP whilst retaining the structuraland functional aspects of the Hsp70 chaperone. Thus, the rHsp70-W90Fmutant unexpectedly provided us with an invaluable tool to further testwhether the direct interaction between Hsp70 and BMP endows Hsp70 withits lysosome protective attributes. Indeed, the rHsp70-W90F mutant hadcompletely lost its ability to protect the lysosomal membranes againstphoto-oxidation and cells against cisplatin-induced lysosomal celldeath, whilst the rHsp70-W580F mutant showed the same efficacy as thewild-type protein. Also the rHsp70-OPBD mutant that showed an unchangedcapacity to bind to BMP rich membranes had lost its ability to protectagainst photo-oxidation and cisplatin. These findings demonstrate thatthe binding of Hsp70 to BMP is required but not sufficient to endow thelysosomal membranes with protection. In addition, an intactcarboxy-terminal peptide-binding domain is necessary for thestabilization of lysosomal membranes in living cells.

Hsp70 inhibitors have for long been considered as interestinganti-cancer drugs. Attention has, however, concentrated on inhibitingthe cytosolic Hsp70, and problems regarding drug-delivery and lack ofspecificity among the Hsp70 family members have presented impassablebarriers for the development of suitable Hsp70 antagonists. Havingestablished that both the binding to BMP and an intact peptide-bindingdomain are required for the cytoprotective effect of Hsp70, and havingverified the potential in targeting Hsp70-BMP interaction, the inventorsnext tested whether the protective effect of the endo-lysosomal Hsp70could also be counteracted by inhibitors of Hsp70 chaperone activity.This was accomplished by incubating the cells with an apoptosis inducingfactor-derived peptide (ADD70), which inhibits the chaperone function ofHsp70 by binding to its peptide-binding domain. It should be noted thatthis large peptide (388 amino acids) does not cross the plasma membrane,and thereby it provided us with another tool to specifically target theendo-lysosomal Hsp70. Notably, incubation of cells with ADD70 peptidecompletely blocked the lysosome-protective effect of endocytosed rHsp70in U-2-OS cells. In order to test whether ADD70 could also counteractthe cytoprotective effect of cells own Hsp70, the inventors investigatedits effect on cisplatin-induced cytotoxicity in Hsp70 transgenicimmortalized murine embryonic fibroblasts (iMEFs), in which thetransgenic Hsp70 confers almost complete resistance againstcisplatin-induced cell death. Remarkably, ADD70-treatment ofHsp70-transgenic iMEFs effectively abolished the Hsp70-mediatedprotective effect and rendered them as sensitive to cisplatin as wildtype iMEFs. The wild type iMEFs express very low levels of Hsp70, andthus the inability of ADD70 to further sensitize them to cisplatinsupports the idea that ADD70-mediated sensitization is, indeed, due tothe inhibition of Hsp70. Akin to anti-BMP treatment, also ADD70treatment sensitized PC-3 and DU-145 prostate carcinoma cells tocisplatin-induced cytotoxicity.

The data presented herein show that Hsp70 interacts directly with theendo-lysosomal anionic phospholipid BMP and that this interactionstabilizes endo-lysosomal membranes. Because the concentration of BMPincreases in endocytic vesicles as the endosomes mature to formmultivesicular bodies, late endosomes and lysosomes, the pH-regulationmight be the way by which Hsp70 is targeted to BMP and lysosomes. Hsp70subdomains differ markedly in their pI values, the ATPase domain having1.72 units higher pI than the peptide-binding domain. Thischaracteristic suggests that at acidic pH, the ATPase domain ispreferentially positively charged, which could facilitate itsinteraction with anionic lipids. As the pH is lowered during theendocytic maturation, the positive charge would build up and any anionicinteraction would be enhanced even further. The data presented hereindemonstrating the dependence of Hsp70-BMP interaction on acidic pH andthe ATPase domain support this theory. Furthermore, molecular modelingof the electrostatic surface of the ATPase domain of Hsp70 revealed thatit forms an almost wedge-like structure with a predominantly positivecharge at the bottom of the wedge even at pH 7.0. Interestingly, W90lies within this positively charged domain, which might give clues towhy the Hsp70-W90F mutation has such a profound impact on the ability ofHsp70 to interact with BMP and stabilize lysosomes. BMP is localizedexclusively in the inner membranes of the endo-lysosomal compartment,where it supports disintegration and lipid extraction from lipidvesicles by acid sphingomyelinase and sphingolipid activator proteinsgiving rise to metabolites such as ceramide and sphingosine-1-phosphate,which have been implicated in destabilization of membranes and celldeath. It should be noted that lysosomal inner membranes can be reachedby invagination of the perimeter membranes at the level of early andlate endosomes, and, therefore, the respective vesicles are likely toalso contain Hsp70. Accordingly, Hsp70 may interfere with BMP's role asa cofactor for sphingolipid hydrolysis and thereby alter the lipidcomposition of the lysosomes. In order to test this hypothesis, theinventors are presently developing mass-spectroscopy based technologyfor quantification of lysosomal sphingolipid metabolites.

Accumulating data suggest that increased expression and alteredtrafficking of lysosomal proteases may form an “Achilles heel” for tumorcells by sensitizing them to lysosomal membrane permeabilization.Therefore, the BMP-Hsp70 interaction on the endo-lysosomal membranes andthe resulting stabilization of the endo-lysosomal compartment providethe cancer cells with protection against this otherwise direct route tocell death. The molecular mechanism underlying this cytoprotectiveeffect now opens new exiting possibilities for sensitization of cancercells to agents that induce lysosomal cell death pathways via specificinhibition of the lysosome stabilizing function of Hsp70. Vice versa,the interaction between Hsp70 and BMP might provide new treatmentstrategies relying on the cytoprotection offered by thelysosome-stabilizing function of exogenously administered Hsp70 forinsults as diverse as pancreatitis, motor and sensory nerve lesions andlight-induced retinal damage.

Materials and Methods

Cell Culture and Reagents

Human U-2-OS osteosarcoma cell lines were cultivated in RPMI 1640(Invitrogen) supplemented with 6% heat-inactivated calf serum andpenicillin-streptomycin. Hsp70 transgenic and appropriate control iMEFswere generated and maintained as described previously (Nylandsted etal., 2004). All cells were grown at 37° C. in a humidified airatmosphere with 5% CO₂ and repeatedly tested and found negative formycoplasma.

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich(Sigma-Aldrich Denmark A/S).

Recombinant Proteins Recombinant Hsp70 and its mutants were generatedusing the pET-16b vector system (Novagen) with induction of proteinexpression and subsequent Ni²⁺-affinity-purification optimized accordingto the manufacturer's protocol.

Labeling of rHsp70 with Alexa Fluor 488 was done according tomanufacturers protocol (Molecular Probes).

Cellular Uptake of Recombinant Proteins and Antibodies:

Sub-confluent cells were cultivated in RPMI 1640 (Invitrogen)supplemented with 6% heat-inactivated calf serum andpenicillin-streptomycin. Recombinant proteins or reticulocyte lysateswere added directly to the medium to obtain the final concentration. Thecells were then grown another 20 h in presence of the protein/lysate.

Loading of cells with an antibody towards BMP (LBPA) (6C4) was doneaccording to techniques in the art.

Quantification of endocytosed rHsp70* was done by growing cells 20 h inthe presence of rHsp70* after which the cells where harvested, washed 3times in PBS and counted. For whole cell uptake 1*10⁵ cells where used.The cells where lysed by incubation for 30 min on ice in 100 μLdigitonin-PBS (200 μg/mL). Fluorescence was measured on a SpectramaxGemini platereader (Molecular Devices). For light membrane fractions(LMF) a total of 10*10⁶ cells where harvested, washed 3 times in PBS andDounce-homogenized until membrane-breakage reached 90% as determined bytrypan-blue staining. The cells where then subjected tomembrane-fractionation by first clearing away the plasma membrane,nucleus and heavy membrane fractions after which the LMF was harvestedby centrifugation at 17000*g for 20 min. The LMF was then split intwo—the first being kept as the “full” LMF. The second fraction wasfreeze/thawn for 5 cycles in liquid nitrogen to break the membranes andsubsequently centrifuged at 20000*g for 20 min in order to separatemembranes from luminal content. All cell work after harvesting was doneat max. 4° C.

Assays for Lysosomal Integrity and Cell Viability

Sub-confluent U-2-OS cells incubated with 2 μg/ml acridine orange for 15min at 37° C. were washed, irradiated and analyzed in Hanks balancedsalt solution complemented with 3% FCS. Cells for single cell imagingwere selected from 8 pre-defined areas of each well in transmittedlight-mode after which the same cells were immediately visualized andexposed to blue light from USH102 100W mercury arc burner (Ushioelectric) installed in a U-ULS100HG housing (Olympus) for 20 sec.Fluorescence microscopy was performed on Olympus IX-70 invertedmicroscope with a LCPlanF1×20 objective with NA=0.40. Loss of lysosomalpH gradient was quantified by counting the loss of intense red staining.

Apoptosis-like cell death was assessed by staining the cells with 0.05μg/ml Hoechst 33342 (Molecular Probes) and counting cells with condensednuclei in an inverted Olympus IX-70 fluorescent Microscope (Filter U-MWU330-385 nm). For each experiment a minimum of eight randomly chosenareas were counted.

The viability of cells was analyzed by the3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction assay as described previously⁶⁷.

Necrotic cells where quantified by flow cytometry by staining the cellsfor 10 min at 37° C. with 2.5 μM SYTOX Green (Molecular Probes) andhereafter measure positively stained cells by their fluorescenceintensity in the FL1 channel of a flow cytometer (FACSCalibur™; BectonDickinson).

Cells were treated with cisplatin as indicated, cytosolic fractions wereobtained by digitonin treatment and cytosolic cysteine cathepsin(zFRase) and caspase-3-like (DEVDase) activities were determined.

RNA Interference

siRNAs used included one targeting the two genes encoding against Hsp70(HSPA1A and HSPA1B); 5′-GCCAUGACGAAAGACAACAAUCUGU-3′ (Invitrogen) and acontrol Hsp70 siRNA described previously. Oligofectamine (Invitrogen)was used as a transfection agent.

Immunodetection

Primary antibodies used included mouse monoclonal antibodies againstHsp70 (2H9; kindly provided by Boris Margulis, Russian Academy ofSciences, St. Petersburg, Russia), glyceraldehyde-3-phosphatedehydrogenase (GAPDH; Biogenesis), BMP (6C4; (Kobayashi et al., 1998)),LIMP-1 (H5C6; developed by J. Thomas August and James E. K. Hildreth andobtained from the Developmental Studies Hybridoma Bank developed underthe auspices of the NICHD and maintained by The University of Iowa,Department of Biological Sciences, Iowa City, USA), cyt c (clone 6H2.B4,BD PharMingen), SERCA (IID8, Calbiochem), and golgin-97 (CDF4, MolecularProbes). Proteins separated by 10% SDS-PAGE and transferred to anitrocellulose membrane were detected by using indicated primaryantibodies, appropriate peroxidase-conjugated secondary antibodies fromDako, ECL Western blotting reagents (Amersham), and Luminescent ImageReader (LAS-1000Plus, Fujifilm).

Tryptophan Fluorescence Spectra and Liposome 90° Light Scattering

The tryptophan fluorescence spectra (RFI) and liposome 90° lightscattering (RSI) were analyzed in a HEPES buffer (20 mM HEPES, 0.1 mMEDTA, pH 7.4 or 6.0 as indicated) employing LUVs consisting of indicatedlipids essentially as described previously. For the RFI, LUVs were addedin 10 μM aliquots and spectra recorded after a 20 min stabilizationperiod. For the RSI, recombinant proteins were added in 0.12 nmolaliquots.

Surface Plasmon Resonance (BIAcore)

For preparation of LUVs a lipid mixture consisting of 10 mol %sphingomyelin, 50 mol % phosphatidylcholine, 20 mol % cholesterol and 20mol % BMP dissolved in organic solvents, was dried under a stream ofargon and rehydrated in Tris/HCl buffer (pH 7.4) (Kolzer et al., 2004).The mixture was freeze-thawed nine times in liquid nitrogen and then inan incubator at 37° C. After ultrasound bath for 15 min the mixture waspassed 21 times through a polycarbonate membrane with a pore diameter of100 nm. Surface plasmon resonance measurements were performed using aBIAcore 2000 system at 25° C. LUVs (total lipid concentration 0.1 mM)were immobilized on the surface of a L1 sensor chip (BIAcore) in PBS(loading buffer). The running buffer used was sodium acetate buffer (50mM, pH 4.5). As a control, acid sphingomyelinase (0.2 μM, 60 μl inrunning buffer) was injected directly on the liposome surface. Responseunits between 4100 RU-5250RU were obtained. The protein of interest wasinjected in running buffer at a flow rate of 20 μl/min at theconcentrations indicated. After injection a dissociation phase of 10 minwas appended.

Molecular Modeling

Primary structure analysis as well as molecular modeling were done withsoftware available from the Expert Protein Analysis System (EXPaSy)proteomics server of the Swiss Institute of Bioinformatics(http://expasy.org/). Molecular modeling was done on basis of thecrystal structure of the human Hsp70-ATPase domain (pdb code: 1S3X) andthe human Hsc70 substrate binding domain (pdb code: 7HSC) withDeepView-Swiss PDB Viewer. Surface models were based on coulombinteraction at pH 7.0 using a solvent dielectric constant of 80 (H₂O).

Statistical Analysis

Statistical analysis was performed using a two-tailed, paired Student'sT-test in order to evaluate the null-hypothesis. The cut-off level forstatistical significance was set to 5% and all groups of data tested forthe comparability of their variances using an F-test. All statisticswere done on a minimum of n=3 independent experiments.

Discussion

The literature has provided evidence that Hsp70 could be present onplasma membranes of tumor cells, as well as in the endolysosomal system.It was furthermore known that Hsp70 could be released to the bloodstreamduring different stress-inducing events, the most typical being fever,trauma and strenuous exercise, the most intriguing probably being frompsychological stress, although this work was mainly done in the field ofimmunology. The presence of Hsp70-species inside the endolysosomalcompartment had also been described for another member of the Hsp70family; the constitutively expressed Hsc70. The function of Hsc70 atthis location had indeed given name to the process known aschaperone-mediated autophagy.

However, from the literature nothing was known about the molecular basisfor the association of Hsp70 with plasma- and endolysosomal membranes,which lead the inventors to the formulation of this project.

The data presented in Example 1 show that Hsp70 is capable ofinteracting with negatively charged membrane lipids such asphospatidylserine (PS), cardiolipin and bis(monoacylglycero)phosphate(BMP) at neutral pH. Upon mimicking the acidity which can be expected inthe early endolysosomal system (pH 6.0), however, the interactionprofile dramatically changes, and the affinity of Hsp70 for BMP becomes20-fold higher than at neutral pH and almost 9-fold greater than for PS.This Hsp70-BMP interaction was verified in a more elaborate BIAcoresystem, in which the pH was now set to that expected in late endosomesand lysosomes (pH 4.5), the main sites for the majority of cellular BMP.Interestingly, the known BMP-interacting protein; acid sphingomyelinase(aSMase), which rely on BMP as a cofactor, only shows half the affinityfor BMP compared to that of Hsp70, illustrating the high relativeaffinity of Hsp70 to BMP.

The interaction of Hsp70 with PS has also been reported by others, ashas an interaction between mouse Hsp70 and acidic glycoceramides, inwhich the interaction depended on the N-terminal ATPase domain and insome cases also on the peptide binding domain (PBD). However, contraryto the systems employed herein, these findings were done in systemsconsisting of basically only one lipid (90-100% and 100% pure lipid,respectively), not likely to resemble any marginally complex lipidenvironment, which one will expect in the eukaryotic cell. However, theimportance of the N-terminal region of Hsp70 for acidic lipidassociation as shown by Harada et al. is in accordance with theinventors finding, that the interaction of Hsp70 with BMP depends on itsN-terminal ATPase domain. The inventors further show that tryptophan 90(W90) of Hsp70 is a critical amino acid as its mutation significantlyreduces the Hsp70-BMP interaction. A hypothetical model argues thatHsp70 contain specific binding sites for the hydrophilic and -phobicparts of acidic glycolipids both in the ATPase as well as in thepeptide-binding domain (PBD).

Although this model might be applicable for Hsp70 binding to acidicglycolipids, the inventors would rather suggest another model for theHsp70-BMP interaction. Based upon the data presented herein that; I) thePBD is only capable of much weaker interactions with BMP; II) theimportance of W90; III) the binding properties of the ATPase domain; andIV) the molecular modeling of surface electrostatic potential of Hsp70,the inventors suggest that Hsp70 interacts with BMP via anelectrostatically positively charged, wedge-like, sub-domain at thebottom of the ATPase cleft. As conservative mutation of W90 tophenylalanine significantly reduces the Hsp70-BMP interaction withoutaffecting the refolding- or ATPase activities of Hsp70, and since thissingle amino acid mutation does not change the electrostatic profile, itis possible, however, that an intermediate of the two models is moreappropriate in explaining the interaction of Hsp70 with a more commonanionic lipid motif. In such a model, the positive surface charge couldfacilitate electrostatic interactions and particular residues such asW90 might be involved in determining specificity of binding of anioniclipid binding partners—in this case, BMP. Interestingly, this couldpotentially implicate Hsp70 as a more general regulator of lipidhomeostasis in the cell. Supporting this are data demonstrating that thelipid membranes of cells might serve as the primary sensors of stresssuch as fever and oxidative stress and hence as the initial inducers ofthe stress response. In face of stress, one could argue that the lipidmembranes of the cell would be crucial compartments to keep inhomeostasis or indeed modify in order to trigger specific signalingevents as a response to the cellular challenge. The binding of Hsp70 tolipids such as BMP and the following increased stability of lysosomalmembranes and perhaps other cellular lipid events could thus represent apart of a general cellular stress response. In the case of cancer such aresponse might have been hi-jacked to serve the cancer's own end, butalso from a broader evolutionary perspective, a coordinatedprotein-lipid response in the face of cellular stress would make goodsense.

The data presented herein showing that only Hsp70, not Hsc70 andHsp70-2, are capable of directly protecting lysosomal membranes arguethat a potential lipid stress response might be specifically regulatedby the major stress-induced Hsp70 itself and not other Hsp70-species.However, as is also shown, depletion of Hsp70-2 also leads to lysosomalmembrane permeabilization and cell death, although in this case thepathway is indirect as it depends on LEDGF. The mechanism for how LEDGFaffects the lysosomal membranes remains unresolved, however.

In order to validate the in vivo relevance of the Hsp70-BMP interaction,the inventors targeted BMP by endocytosed antibodies and lysosomal Hsp70by endocytosis of the otherwise cell-impermeable AIF-derived polypeptideADD70. This verified that the interaction between Hsp70 and BMP servesto stabilize lysosomal membranes as cells subsequently wheresignificantly sensitized to the effects of direct lysosomal membranedisruptive stimuli as well as the LMP-inducing chemotherapeutic agentcisplatin, the programmed cell death-profile of which was characterizedas part of this project. Expression of ADD70 has formerly been shown tosensitize cancer cells to a variety of death stimuli and decrease thetumorigenicity of rat colon carcinoma and mouse melanoma cells insyngeneic animals. The major difference between this approach and theapproach presented herein is that the present inventors sought tospecifically target the lysosomal Hsp70 through endocytosis of ADD70,whereas the former studies utilized cytosolic expression of ADD70 inorder to target the more abundant cytoplasmic Hsp70. The success intargeting the endolysosomal Hsp70-BMP interaction also provided acertain proof-of-concept of the idea of targeting lysosomal componentsthrough endocytosis for therapeutic means, a concept which could havebroad therapeutic implications, as one could imagine sensitizing e.g.cancer cells to agents that induce lysosomal cell death pathways viaspecific inhibition of the lysosome stabilizing function of Hsp70. Viceversa, the interaction between Hsp70 and BMP might provide new treatmentstrategies relying on the cytoprotection offered by thelysosome-stabilizing function of exogenously administered Hsp70 forinsults as diverse as pancreatitis, motor and sensory nerve lesions andlight-induced retinal damage. Indeed, the concept of utilizing theendocytic machinery for introduction of specific cytotoxic compoundshave already been explored, as endocytic delivery of ahydrocarbon-stapled BH3 helix based on the pro-apoptotic BH3 interactingdomain death agonist, Bid, was shown to induce apoptosis in leukemiacells. This process depended on the BH3 helix leaving the endocyticcompartment intact and activating Bax and Bak in order to inducecytochrome c release and activate a mitochondrial program of apoptosis.However, the mechanism of escape from the endocytic system wasunfortunately not addressed in this paper.

As shown herein, the interaction between Hsp70 and BMP depends on theATPase domain of Hsp70. Interestingly, recent reports on the Hsp70cochaperone, Hsp70 binding protein 1 (HspBP1), might emphasize theimportance of this positively charged area of Hsp70. A study of thecrystal structure of HspBP1 complexed with part of the ATPase domain ofHsp70 has revealed that the interaction between these two was mediatedby a curved, all a-helical fold in HspBP1 containing four armadillo-likerepeats. The concave face of this curved fold embraces lobe II of theATPase domain, the same lobe which forms the major part of theelectrostatically positively charged volume of Hsp70s ATPase domain,which the inventors argue mediate the interaction between Hsp70 an BMP.A further perspective on this is provided by a another study, in which14 cancer cell lines were characterized with regard to their relativeHsp70/HspBP1 levels. This other study found that cell lines with a highHspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs thanthose with low ratio and that overexpression of HspBP1 promotedlysosomal membrane permeabilization. Based on these reports, and thedata presented in this Example, one could argue for a model in whichHspBP1 by binding to the positively charged area of the ATPase domain ofHsp70, disrupts its interaction with BMP and hence its stabilizingeffect on endo-lysosomal membranes, resulting in increased sensitivityto LMP-inducing stimuli. As such, the armadillo-repeat domain of HspBP1could potentially form the basis of an intelligent drug design, much asthe case for ADD70. The efficacy of such HspBP1-derived molecules wouldbe easy to test in the systems described herein and presents aninteresting path towards further applications of the molecular mechanismdescribed herein.

As the inventors show herein, Hsp70 binds with high affinity to BMP atacidic pH 4.5, even almost 2-fold higher than what is the case for the“classical” BMP binding partner acid sphingomyelinase (aSMase).Interestingly, BMP serve as a stimulatory cofactor for enzymatichydrolysis of not only sphingomyelin via aSMase, but of mostmembrane-bound sphingolipids as it also functions as a cofactor forsphingolipid activator proteins (SAPs/Saposins) A-D. An obvious questionwould thus be, whether Hsp70 by its binding to BMP somehow alters thebinding properties of aSMase and the Saposins, hereby modifying thecatabolism of membrane sphingolipids and glycosphingolipids and thegeneration of downstream effector molecules such as ceramide and itsmetabolites, Ceramide-1-phosphate, sphingosine andsphingosine-1-phosphate, all of which have been implicated in both cellsurvival and death. Indeed, the inventors have found that Hsp70 iscapable of modulating the binding of aSMase to BMP-containinglipososomes at pH 4.5, depending on the concentration of Hsp70. As canbe seen, low concentrations (3-150 nM) of Hsp70 facilitate theinteraction of aSMase with BMP-Hsp70 liposomes, whereas higherconcentrations of Hsp70 (300-1500 nM) has the opposite effect. Althoughour working concentration in the medium when Hsp70 is added forendocytosis is 300 nM, it would be hard to estimate a givenintralysosomal concentration on this basis and any conclusions as towhat effect Hsp70 might have on aSMase activity in vivo would remainspeculative. However, staining of the Hsp70-transgenic (Hsp70-TG) andwildtype (WT) iMEFs with a monoclonal antibody against ceramide revealedthat the Hsp70-transgenic mice show a clear upregulation of ceramide,which is present in a characteristic beads-on-a-string pattern in theperipheri of the cells as well as near the nucleus. Further analysis ofthe ceramide-profile of the iMEFs via lipid extraction and subsequentlipid mass spectroscopy has confirmed these findings, as the cumulativelevels of ceramide were increased from an average 10.2 ng ceramide/mgprotein for the iMEF-WT to 14.9 ng/mg for the Hsp70 transgenic iMEFs.The inventors have further substantiated that this effect can beascribed to the action of Hsp70, as the inventors have also profiled ourU-2-OS cells loaded with rHsp70 (i.e. 300 nM rHsp70 in full media for 24h, analogous to all other Hsp70-endocytosis experiments presentedherein). The quantification of ceramide in Hsp70-loaded U-2-OS cellsshowed an increase in cumulative levels of ceramide from 2.99 ngceramide/mg protein for the control cells to 5.10 ng/mg for theHsp70-loaded cells (the experiment has only been done once at the timeof writing). However, taken together they all support a role for Hsp70in modulating ceramide levels in cells, although further validation ofcourse is needed. Yet, if these data can be verified, a series ofquestions present themselves, such as the compartmentalization of theceramide species, quantification of specific ceramide-species (of whichthere is at least 50 distinct molecular species), profiling of ceramidelevels in the face of various stresses, transformation status of cellsetc.

Interestingly, a previous study has addressed one of these questions,which show that heat shock (42.5° C. for 2 h) causes the accumulation ofceramide in Molt-4 acute leukemic lymphocytes. This accumulation couldbe blocked by the pharmacological inhibitors Fumonisin B1 and myriocin,the latter of which is regarded as a specific inhibitor of the de novopathway of ceramide synthesis as it blocks the action of serinepalmitoyltransferase, the enzyme which initiates the de novo synthesisof new sphingolipids from serine and palmitoyl-CoA. A partial mechanismfor this increase in de novo synthesis of ceramide has been described inyeast, in which heat stress induces an acute influx of serine into theER that drives de novo synthesis. It will be interesting to test if theincrease in ceramide levels observed upon endocytosis of rHsp70 can bemodulated by these pharmacological inhibitors or whether the observedincreases stem from the catabolic pathways of sphingolipid degradationand the stimulation of these by Hsp70 binding to BMP. Of course, acompound model could also be hypothesized. In this model, an initialheat stress could lead to membrane fluidization, serine influx and rapidinitiation of de novo sphingolipid synthesis. Subsequently, theinduction of Hsp70, as a consequence of the heat stress, would lead toincreased Hsp70 levels in the cell, Hsp70-interaction with BMP, increasein aSMase-activity—and possibly also SAP activity—resulting in thegeneration of ceramide by the catabolic pathways. This secondaryresponse could either complement the initial de novo induction or maybetake over for it as the continuous de novo response would rely on acontinuous supply of serine and palmitioyl-CoA. It remains however, tobe tested if the cellular protection is a consequence of the increase inceramide itself or maybe should be contributed to altered levels of itsupstream and downstream metabolites.

At this point, some major questions remain to be answered—How does Hsp70end up in the extracellular milieu and inside the endolysosomalcompartment? Is Hsp70 secreted and then taken up by endocytosis?—or isit present inside lysosomes or more specialized secretory lysosomes,waiting for a release signal in the form of stress? And perhaps moreimportantly—what is the biological significance of the presence of Hsp70in the extracellular environment?

Although the work presented in this work is not capable of answeringthese complex questions, some deductions can however be made. First,Hsp70 could be endocytosed in all cell lines tested in this project,arguing for a common way of recognizing extracellular Hsp70 (eHsp70).This is in accordance with data showing that eHsp70 can bind to a numberof receptors on different leucocyte sub-populations. The receptorsinvolved in extracellular Hsp70 (eHsp70) recognition mainly includepattern recognition receptors (PRRs) and consist of a variety ofreceptors from different receptor families such as the toll likereceptors (TLR), scavenger receptors and c-type lectins. As the work inthis project has not addressed by which initial mechanism Hsp70 isendocytosed (receptor-mediated, raft-dependent, clathrin-dependent etc.)it cannot be said whether PRRs are responsible for the endocytosis ofeHsp70 seen in our systems. However, 10-fold excess of un-labelled Hsp70could not compete with AF488-labelled Hsp70 uptake in neither U-2-OScells nor iMEFs—on the contrary, endocytosis was significantly enhancedin the presence of excess un-labelled Hsp70, which to some extent arguesagainst a saturable mechanism of uptake.

The focus of the immunological field has mainly been on the cytokineresponse and activation of the innate immune defense elicited by eHsp70binding to the PRRs and hence not much regard has been given to theeffect of eHsp70 after receptor binding and initiation of signaling.

The release mechanisms of Hsp70 into the extracellular milieu and theeffects of Hsp70 once herein have to some extent been addressed,although a satisfying molecular insight into these exciting mechanismsis still lacking. Nevertheless, plenty of evidence exists for thepresence of Hsp70 in the circulatory system after stress andaccumulating data support a role for eHsp70, whether stress-induced orexogenously delivered, in neuroprotection as well as in priming theprimary immune defense system.

With regard to release of Hsp70, the first evidence for the transfer ofHsp70 from one cell to another came from studies in the squid giantaxon, and during the reproduction of these results in cultured ratembryo cells, evidence was presented that a non-classical pathway ofexocytosis could be responsible for the release of Hsp70.

It has been suggested that Hsp70 along with other heat shock proteinsare only released under pathological circumstances resulting in necroticdeath and not during programmed cell death. Recent studies however, haveshown that Hsp70 can be released from intact cells by active mechanismsand that the degree of stimulus determines the mode of release.Importantly, no known studies have reported a direct correlation betweeneHsp70 and markers of muscle damage although major increases of eHsp70can be detected in the peripheral bloodstream upon physical exercise.Most convincing, and perhaps also most intriguing, are the discoveriesshowing that psychological stress such as predatory fear and electricshock can evoke a stress induced eHsp70 release, a process which wassuggested to be dependent on cathecholamine signaling. This isparticularly interesting as catecholamines via the 0 i-adrenergicreceptor can lead to intracellular calcium-fluxes, and calcium-fluxescan cause exocytosis of exosomes, multivesicular bodies and lysosomes.As such, during times of stress, increases in noradrenaline acting uponE I-adrenergic receptors could result in a calcium flux within the cellwith the subsequent release of Hsp70 within exosomes. Evidence for thishypothesis comes from demonstrations that eHsp70 can be released invesicles characterized as exosomes, but evidence has also been presentedthat eHsp70 can be released as free eHsp70, both in cellular systems aswell as in vivo. It has also been suggested that lipid rafts are neededfor eHsp70 release although this has also been disputed. Moreover, ithas been shown that a functional lysosomal compartment is necessary forrelease of eHsp70 and that this release is accompanied by the presenceof lysosomal marker proteins on the surface of the cells, suggesting asecretion dependent on plasma- and lysosomal membrane fusion.

Regardless of whether the released Hsp70 is present in exosomes or asfree eHsp70, it is interesting to note that some sort of secretoryMVB/late endosomal/lysosomal compartment is apparently involved in allmodes of release. Based upon these data, and the results obtainedherein, a more complex hypothesis for how Hsp70 escapes from the cytosolto the extracellular environment can be formulated. The release of Hsp70would still depend on increases in intracellular calcium, as this wouldserve as the signal for exocytosis of endo-lysosomes. The presence ofHsp70 within this compartment would however be dependent on theinteraction of Hsp70 and BMP as described herein, as Hsp70 would beeffectively aggregated on BMP-containing inner membranes in lateendosomes/MVBs/lysosomes. Hsp70 could either arrive in late endosomesand lysosomes from extracellular uptake such as endocytosis as alsodescribed herein, or through invaginations of the perimeter membranes ofearly and late endosomes as well as lysosomes, which would bringintracellular Hsp70 and BMP in proximity. The acidity of the compartmentwould maintain a strong preference for Hsp70s localization toBMP-containing membranes. Upon exocytosis, some Hsp70 would still bebound to BMP-containing exosomes, but the neutral pH encountered in theextracellular environment would now favour an Hsp70-BMP equilibriumshifted significantly towards more unbound Hsp70, resulting in both freeas well as exosome-bound Hsp70, which could then exert theirextracellular functions.

In summary, the data presented herein show that Hsp70 interacts directlyand pH-dependently with the endo-lysosomal anionic phospholipid BMP. Theinventors demonstrate that the binding of Hsp70 to BMP is mediated viaHsp70s ATPase domain, involving tryptophan 90, and that this interactionresults in the stabilization of endo-lysosomal membranes, possibly byinfluencing the activity of other BMP-binding proteins. The inventorsalso show that the elucidation of this molecular mechanism opens newexiting possibilities for sensitization of cancer cells to agents thatinduce lysosomal cell death pathways via specific inhibition of thelysosome stabilizing function of Hsp70. Vice versa, the interactionbetween Hsp70 and BMP might provide new treatment strategies relying onthe cytoprotection offered by the lysosome-stabilizing function ofexogenously administered Hsp70.

Example 2: Interaction Between Hsp70 and Bis(Monoacylglycero)PhosphateActivates Acid Sphingomyelinase, Stabilizes Lysosomal Membranes andPromotes Cell Survival

Heat shock protein 70 (Hsp70) is an evolutionarily highly conservedmolecular chaperone that promotes the survival of stressed cells byinhibiting lysosomal membrane permeabilization, a hallmark ofstress-induced cell death. Clues to its molecular mechanism of actionmay lay in the recently reported stress- and cancer-associatedtranslocation of a small portion of Hsp70 to the lysosomal compartment.Here, we show that Hsp70 stabilizes lysosomes by enhancing the activityof acid sphingomyelinase (ASM), a lysosomal lipase that hydrolyzessphingomyelin to ceramide and phosphorylcholine. In acidic environmentHsp70 binds with high affinity and specificity to an endo-lysosomalanionic phospholipid bis(monoacylglycero)phosphate (BMP), an essentialco-factor for ASM, thereby facilitating the binding of ASM to BMP andstimulating ASM activity. The inhibition of the Hsp70-BMP interaction byBMP antibodies or a point mutation (W90A) in Hsp70 as well as theinhibition of ASM activity by desipramine effectively revert theHsp70-mediated stabilization of lysosomes. Notably, the reduced ASMactivity in cells from patients with Niemann-Pick disease A (NPDA). asevere lysosomal storage disorder caused by mutations in the ASM gene,is also associated with a dramatic decrease in lysosomal stability, andthis phenotype can be effectively corrected by restoring the lysosomalASM activity by treatment with recombinant Hsp70 or ASM. Taken together,these data open exciting possibilities for the treatment of lysosomalstorage disorders and cancer with non cell permeable compounds thatenter the lysosomal lumen via the endocytic delivery pathway.

Lysosomal proteases, cathepsins, are important effectors inevolutionarily conserved cell death programs induced by a wide varietyof stresses. Cathepsin-dependent cell death is characterized by an earlylysosomal membrane permeabilization and subsequent translocation ofcathepsins into the cytosol, where they can initiate caspase-dependentand -independent cell death pathways. In order to test whether thelysosomal localization is crucial for the reported ability of Hsp70 tostabilize lysosomal membranes and protect cells against stress-inducedcell death, we took advantage of the endocytic machinery of cells totarget recombinant Hsp70 (rHsp70) into the lysosomes. Immunocytochemicaland biochemical analysis of U-2-OS osteosarcoma cells incubated withfluorochrome-labeled rHsp70 revealed effective uptake of rHsp70, itsspecific co-localization with late endosomal and lysosomal markers andbinding to lysosomal membranes (FIG. 5a,b and FIG. 9). Using a real timeimaging to monitor lysosomal membrane integrity (FIG. 5c ), we showedthat the endocytosed rHsp70 protected lysosomes against photo-oxidation(FIG. 5d ). Furthermore, a short interfering RNA (siRNA) specific forHsp70 sensitized the lysosomes to photo-oxidation, and this effect wasfully reverted by endocytosed rHsp70 aptly demonstrating that theprotective effect of endogenous Hsp70 is mediated by the small fractionof the protein in the lysosomal lumen (FIG. 5e ). In spite of similaruptake (data not shown), no lysosomal stabilization was observed withrecombinant Hsc70 and Hsp70-2, which share 86% and 84% amino acidsequence homology with Hsp70, respectively (FIG. 5d ).

The presence of Hsp70 in the lysosomal membranes and its ability tosurvive the hydrolytic lysosomal environment suggest that it binds tothe lysosomal membrane lipids. Thus, we investigated the interaction ofHsp70 with palmitoyl-oleoyl-phosphatidylcholine (POPC) large unilamellarvesicles (LUVs) containing various membrane-associated anionic lipids,i.e. palmitoyl-oleoyl-phosphatidylserine (POPS; primarily in plasmamembrane), cardiolipin (primarily mitochondrial) and BMP (primarily inlate endosomes/lysosomes). Taking into account the increasingly acidicmilieu of the endo-lysosomal compartment upon maturation to lysosomes,we compared the protein-lipid interactions in neutral (pH 7.4) andacidic (pH 6.0) conditions (FIG. 6a ). At pH 7.4, rHsp70 caused a littlerelative change in the 90° light scattering in POPC liposomes indicatinga very weak binding. As reported earlier for POPS, all negativelycharged lipids enhanced the binding of rHsp70 to the liposomes atneutral pH approximately 4-fold irrespective of the charge density onthe liposome surface (ranging from −1 to −2) (FIG. 6a ). Remarkably, thebinding to BMP was almost 20 times stronger at the acidic pH as comparedto the neutral pH, whereas the binding to POPS was only slightlyincreased upon acidification (FIG. 6a ). The high affinity binding ofHsp70 to BMP in acidic pH was confirmed in an independent set of BIAcoreexperiments (FIGS. 6e and 7a ). Importantly, BMP antibodies delivered tothe endo-lysosomal compartment by endocytosis effectively inhibited theability of rHsp70 to stabilize the lysosomes in living cells (FIG. 6b ),and sensitized the cells to cisplatin (FIG. 6c ), an anti-cancer drugthat induces lysosomal leakage.

In order to investigate which part of the Hsp70 protein is responsiblefor the BMP binding, we measured the fluorescence shift of thetryptophans upon docking of rHsp70 and its mutants into BMP-containingliposomes. The loss of signal in relative peak fluorescence intensityfor the Hsp70 mutant lacking amino acids 119-426 in the amino-terminalATPase domain (rHsp70-□ATP), but not for that lacking amino acids437-617 in the carboxy-terminal peptide-binding domain (rHsp70-□PBD),indicated that the ATPase domain was required for the high affinitybinding of Hsp70 to BMP (FIG. 6d ). Next, we substituted the twotryptophans in Hsp70 with phenylalanines (W90F and W580F) and studiedwhich tryptophan is responsible for the fluorescence shift induced bylipid binding. The reduction of the signal only with rHsp70-W90Findicated that the NH₂-terminus of the protein docked into the lipidlayer (FIG. 6d ). A more quantitative BIAcore analysis of the BMP-rHsp70interaction confirmed that Hsp70 interacted with BMP mainly through itsATPase domain (FIG. 6e ). Surprisingly, the W90F mutation specificallyabolished the interaction between rHsp70 and BMP whilst retaining thestructural (folding as analyzed by far- and near-UV circular dichroism)and functional (luciferace folding and ATP hydrolysis) aspects of theHsp70 chaperone (FIG. 6e and data not shown). Thus, the rHsp70-W90Fmutant provided us with an invaluable tool to further test whether thedirect interaction between Hsp70 and BMP endows Hsp70 with its lysosomeprotective attributes. Indeed, the rHsp70-W90F mutant had completelylost its ability to protect the lysosomal membranes againstphoto-oxidation and cells against cisplatin-induced lysosomal celldeath, whereas the rHsp70-W580F mutant showed the same protective effectas the wild-type protein (FIGS. 6f and g ). Importantly, mutant Hsp70proteins were endocytosed essentially as effectively as the wild typeHsp70 (data not shown). Thus, we conclude that the binding of Hsp70 toBMP is essential for the lysosome stabilizing effect of Hsp70.

Because the concentration of BMP increases in endocytic vesicles as theendosomes mature to form lysosomes, the pH-regulation might be the wayby which Hsp70 is targeted to lysosomes. Calculations (PROTPARAM, EXPaSyproteomics server, Swiss Institute of Bioinformatics) revealed that theATPase domain of Hsp70 has 1.72 units higher theoretical pI than thepeptide-binding domain (6.62 vs. 4.9). This characteristic suggests thatat acidic 20 pH, the ATPase domain is preferentially positively charged,which could facilitate its interaction with anionic lipids. Our datademonstrating the dependence of Hsp70-BMP interaction on acidic pH andthe ATPase domain support this theory. Furthermore, molecular modelingof the electrostatic surface of the ATPase domain of Hsp70 revealed thatit forms an almost wedge-like structure with a predominantly positivecharge at the bottom of the wedge containing W90 possibly explaining theprofound impact of W90F mutation on the ability of Hsp70 to interactwith BMP and stabilize lysosomes (FIG. 6h ).

BMP binds ASM with high affinity and stimulates its ability to hydrolyzesphingomyelin to ceramide and phosphorylcholine. The BIAcore analysisrevealed that pretreatment of the BMP-containing LUVs with rHsp70 atsub-equimolar concentrations facilitated the subsequent binding of ASM,whereas higher rHsp70 concentrations showed an inhibitory effect (FIGS.7a and 10). Remarkably, Hsp70 transgenic murine embryonic fibroblasts(Hsp70-MEFs), which are protected against stress-induced lysosomaldamage (FIG. 7e ), displayed significantly higher ASM activity than wildtype MEFs (WT-MEFs), and the treatment of WT-MEFs with rHsp70 at acytoprotective concentration (300 nM) increased the ASM activity to thelevel comparable to that in Hsp70-MEFs (FIG. 7b ). In order to testwhether ASM is responsible for the lysosome stabilizing effect wetreated the cells with desipramine, a well characterized pharmacologicalASM inhibitor. Desipramine reduced the viability of MEFs in adose-dependent manner and the cell death was associated with a massivepermeabilization of lysosomes as demonstrated by the leakage oflysosomal cathepsins into the cytosol (FIGS. 7c and d ). Notably,desipramine-induced cell death and lysosomal leakage were significantlyreduced in Hsp70-MEFs as compared to WT-MEFs. Furthermore, inhibition ofASM with subtoxic concentration of desipramine reverted the lysosomalstress resistance of Hsp70-MEFs to the level of WT-MEFs as evidenced byaccelerated loss of lysosomal membrane integrity upon photo-oxidation(FIG. 7e ). The lysosome protective role of ASM was further supported bydata showing that lysosomes in fibroblasts from patients with NPDA, afatal lysosomal storage disorder caused by mutations in the ASM gene,displayed extreme sensitivity to photo-oxidation-induced damage (FIG. 8a). Remarkably, rHsp70 was also capable of enhancing the enzymaticactivity of the endogenous mutated ASM as well as the simultaneouslyloaded rASM in the patient cells (FIG. 8b ). The increased ASM activityobtained by loading the lysosomes with rHsp70, rASM or the combinationof the two correlated with their ability to stabilize the lysosomes andto normalize the volume of the dramatically enlarged lysosomalcompartment in NPDA cells (FIG. 8b-d ). It should be noted that akin torHsp70, also rASM localized to the lysosomes (FIG. 8b ).

Taken together our data indicate that Hsp70-BMP interaction stabilizeslysosomes via a mechanism involving the regulation of sphingomyelinmetabolism rather than direct physical stabilization of the membrane.Such an indirect effect is supported by the fact that BMP is localizedexclusively in the inner membranes of the endo-lysosomal compartment,where its major function is to support the disintegration and lipidextraction from lipid vesicles by ASM and sphingolipid activatorproteins. Interestingly, ASM-mediated increase in lysosomal ceramideconcentration modifies the steric conformation of lysosomal membranesand thereby facilitates their fusion with other intracellular vesiclesand plasma membrane. Thus, the changes in the lysosomal membranecomposition and volume as a result of the ceramide-induced enhancedfusion capacity may contribute to the Hsp70-mediated increase inlysosomal stability. On the other hand, various apoptotic stimuli inducethe translocation of ASM to the outer leaflet of plasma membrane, whereceramide can form lipid microdomains that function as sites foractivation of membrane-associated signaling molecules involved inapoptotic signaling. Thus, ceramide may have opposing effects on cellsurvival depending on whether it is produced inside the lysosome or onthe plasma membrane.

The above-described molecular mechanism underlying the cytoprotectiveeffect of Hsp70 opens new exiting possibilities for sensitization ofcancer cells to agents that induce lysosomal cell death pathways viaspecific inhibition of the lysosome stabilizing function of Hsp70. Viceversa, the ability of exogenously administered rHsp70 alone or incombination with rASM can be directly challenged as a novel treatmentfor NPD patients, whose therapeutic options are currently limited togene and stem cell therapies.

Methods Summary

WT- and Hsp70-MEFs were generated, immortalized and maintained asdescribed in the art. Human NPDA fibroblasts (83/24) originate from askin biopsy of a 5 month old patient with hepatosplenomegaly.Recombinant proteins were generated using the pET-16b vector system andNi²⁺-affinity-purification (Novagen), and labeled with Alexa Fluor 488according to manufacturers protocol (Molecular Probes). To analyze thelysosomal integrity, we developed a real time imaging method of cellsstained with acridine orange, a metachromatic weak base that accumulatesin the acidic compartment of the cells staining them red and sensitizingthem to photo-oxidation. The photo-oxidation-induced loss of thelysosomal pH-gradient and leakage of acridine orange to the cytosol fromindividual lysosomes was quantified visually as “loss of red dots” inU2-O—S cells and as a decrease in red and an increase in greenfluorescence by Zeiss LSM DUO Software in fibroblasts. The total andcytoplasmic (digitonin-extracted) cathepsin activities were measured indigitonin-treated samples using zFR-AFC (Enzyme System Products) probeas described in the art. The tryptophan fluorescence spectra andliposome 90° light scattering were analyzed in a HEPES buffer (20 mMHEPES, 0.1 mM EDTA, pH as indicated) essentially as described in theart. Surface plasmon resonance measurements were performed withimmobilized LUVs using a BIAcore 2000 system as described in the art.Hsp70 siRNA (5′-GCCAUGACGAAAGACAACAAUCUGU-3′) and a control Hsp70 siRNAwere transfected with Oligofectamine (Invitrogen). Immunodetection wasperformed with standard protocols. Apoptosis-like cell death andlysosomal membrane permeabilization were analyzed essentially asdescribed in the art. ASM activity was analyzed by Amplex RedSphingomyelinase Assay Kit (A12220) from Molecular Probes withmodifications described in the art. Statistical analysis was performedusing a two-tailed, paired Student's T-test and all groups of data weretested for the comparability of their variances using an F-test.

Methods

Cell Culture and Reagents.

Human U-2-OS Osteosarcoma Cells were Cultivated in RPMI 1640(Invitrogen) supplemented with 6% heat-inactivated calf serum andpenicillin-streptomycin. Hsp70 transgenic and appropriate control MEFswere generated and maintained as described in the art. Human primaryNPDA fibroblasts where grown in MEF media further supplemented with 1%Na-Pyruvate, 1% HEPES, 1% L-Glutamine. All cells were grown at 37° C. ina humidified air atmosphere with 5% CO₂ and repeatedly tested and foundnegative for mycoplasma. Unless otherwise stated, all chemicals werepurchased from Sigma-Aldrich (Sigma-Aldrich Denmark A/S).

Assays for Lysosomal Integrity.

Sub-confluent cells incubated with 2 μg/ml acridine orange for 15 min at37° C. were washed, irradiated and analyzed in Hanks balanced saltsolution complemented with 3% fetal calf serum. Cells for single cellimaging were selected from 8 pre-defined areas of each well intransmitted light-mode after which the same cells were immediatelyvisualized and exposed to blue light from USH102 100W mercury arc burner(Ushio electric) installed in a U-ULS100HG housing (Olympus) for 20 sec.Fluorescence microscopy was performed on Olympus IX-70 invertedmicroscope with a LCPlanF1×20 objective with NA=0.40. Loss of lysosomalpH gradient was quantified by counting the loss of intense red staining.A more elaborate method for assaying lysosomal integrity was developedto handle the larger lysosomal compartment of the various fibroblastsused in this study. Cells for single cell imaging were selected from 8pre-defined areas of each well in transmitted light-mode after which thesame cells were immediately and continuously exposed to 489 nm lightfrom a 100 mW diode laser while laser scanning micrographs wherecaptured every 330 ms on a Zeiss LSM LIVE DUO confocal system in twochannels defined by bandpass filters for 495-555 nm (green) and LP650 nm(Red) light. The resulting timelapse movies where subsequently analysedby the integrated Zeiss LSM DUO software. The total and cytoplasmic(digitonin-extracted) cathepsin activities were measured indigitonin-treated samples using zFR-AFC (Enzyme System Products) probeas described in the art.

Assays for Cell Viability.

Cell density was assessed by the3-(4,5-dimethylthiazole-2-y)-2,5-diphenyltetrasodiumbromide (MTT,SIGMA-Aldrich) reduction assay essentially as described in the art.Apoptosis-like cell death was assessed by staining the cells with 0.05μg/ml Hoechst 33342 (Molecular Probes) and counting cells with condensednuclei in an inverted Olympus IX-70 fluorescent Microscope (Filter U-MWU330-385 nm). For each experiment a minimum of eight randomly chosenareas were counted.

Immunodetection and Microscopy.

Primary antibodies used included mouse monoclonal antibodies againstHsp70 (2H9; kindly provided by Boris Margulis, Russian Academy ofSciences, St. Petersburg, Russia), glyceraldehyde-3-phosphatedehydrogenase (GAPDH; Biogenesis), BMP (6C4), lysosomal integralmembrane protein-1 (H5C6; developed by J. Thomas August and James E. K.Hildreth and obtained from the Developmental Studies Hybridoma Bankdeveloped under the auspices of the NICHD and maintained by TheUniversity of Iowa, Department of Biological Sciences, Iowa City, USA).Proteins separated by 10% SDS-PAGE and transferred to a nitrocellulosemembrane were detected by using indicated primary antibodies,appropriate peroxidase-conjugated secondary antibodies from Dako, ECLWestern blotting reagents (Amersham), and Luminescent Image Reader(LAS-1000Plus, Fujifilm). For immunocytochemistry Alexa Fluor®-576- orAlexa Fluor®-488-conjugated secondary antibodies were used. LysotrackerRed® was used for live visualization of the lysosomal compartment.Fluorescence images were taken using a Zeiss Axiovert 100M laserscanning microscope. Lysotracker quantification and timelapse movies forlysosomal integrity were done on a Zeiss LSM LIVE DUO system.

Tryptophan Fluorescence Spectra and Liposome 90 Light Scattering.

The tryptophan fluorescence spectra (RFI) and liposome 900 lightscattering (RSI) were analyzed in a HEPES buffer (20 mM HEPES, 0.1 mMEDTA, pH 7.4 or 6.0 as indicated) employing LUVs consisting of indicatedlipids essentially as described in the art. For the RFI, LUVs were addedin 10 μM aliquots and spectra recorded after a 20 min stabilizationperiod. For the RSI, recombinant proteins were added in 0.12 nmolaliquots.

Surface Plasmon Resonance (BIAcore).

For preparation of LUVs a lipid mixture consisting of 10 mol %sphingomyelin, 50 mol % phosphatidylcholine, 20 mol % cholesterol and 20mol % BMP dissolved in organic solvents, was dried under a stream ofargon and rehydrated in Tris/HCl buffer (pH 7.4). The mixture wasfreeze-thawed nine times in liquid nitrogen and then in an incubator at37° C. After ultrasound bath for 15 min the mixture was passed 21 timesthrough a polycarbonate membrane with a pore diameter of 100 nm. Surfaceplasmon resonance measurements were performed using a BIAcore 2000system at 25° C. LUVs (total lipid concentration 0.1 mM) wereimmobilized on the surface of a L1 sensor chip (BIAcore) in PBS (loadingbuffer). The running buffer used was sodium acetate buffer (50 mM, pH4.5). As a control, acid sphingomyelinase (0.2 μM, 60 μl in runningbuffer) was injected directly on the liposome surface. Response unitsbetween 4100 RU-5250RU were obtained. The protein of interest wasinjected in running buffer at a flow rate of 20 μl/min at theconcentrations indicated. After injection a dissociation phase of 10 minwas appended. In the case where rASM followed rHsp70, rASM was added for180 sec after the 10 min rHsp70-dissociation phase followed by yet a 10min dissociation phase.

Molecular Modeling.

Primary structure analysis as well as molecular modeling were done withsoftware available from the Expert Protein Analysis System (EXPaSy)proteomics server of the Swiss Institute of Bioinformatics(http://expasy.org/). Molecular modeling was done on basis of thecrystal structure of the human Hsp70-ATPase domain (pdb code: 1 S3X) andthe human Hsc70 substrate binding domain (pdb code: 7HSC) withDeepView-Swiss PDB Viewer. Surface models were based on coulombinteraction at pH 7.0 using a solvent dielectric constant of 80 (H₂O).

Statistical Analysis.

Statistical analysis was performed using a two-tailed, paired Student'sT-test in order to evaluate the null-hypothesis. The cut-off level forstatistical significance was set to 5% and all groups of data tested forthe comparability of their variances using an F-test. All statisticswere done on a minimum of n=3 independent experiments.

Example 3: Effect of Benyl Alcohol on Lysosomal Storage Disease

It is shown in Examples 2 and 3 that Hsp70 has a lysosome stabilizingeffect via an interaction with BMP. In order to evaluate if this effectis also observed when exposing cells to a chemical Hsp70 inducer,Niemann-Pick Type A (NPDA) patient fibroblasts were treated with thesmall molecule Hsp70 inducer; Benzyl Alcohol (BA). Results are shown inFIG. 11. First, NPDA cells were treated with increasing doses of BA (0,20, 30, 35, 40, 45 mM), lysed, and analysed by western blotting. Thesame amount of protein was loaded in each well. Hsp70 protein expressionwas evaluated for each condition, and shows that BA induced Hsp70expression in a dose-dependent manner (primary antibody: StressgenSPA-810, specific for Hsp70). Next, the stability of NPDA Götz lysosomesafter treatment with 40 mM BA was evaluated, using the same methods asdescribed in Example 2. An increased lysosomal stability was observed inresponse to BA. Finally, the lysosomal cross-sectional area in NPDA Götzcells after treatment with 40 mM BA was evaluated, using the samemethods as described in Example 2. A decreased pathology is observed.

Items

-   -   1. Method for modulating the enzymatic activity of an enzyme,        wherein said enzyme interacts with BMP, said method comprising        the step of administering Hsp70, or a functional fragment        thereof, in a form suitable for allowing interaction between BMP        and Hsp70, or said functional fragment thereof, and thereby        modulating the enzymatic activity of an enzyme interacting with        BMP.    -   2. Method of item 1, wherein Hsp70 or said functional fragment        thereof forms a covalent or non-covalent complex with BMP.    -   3. Method of any one of the preceding items, wherein BMP        interacts with a saposin.    -   4. Method of item 3, wherein said saposin is selected from the        group consisting of saposin A, saposin B, saposin C, and saposin        D.    -   5. Method of any one of the preceding items, wherein said enzyme        is selected from the group consisting of sphingomyelinase,        acidic sphingomyelinase, sialidase, alpha-galactosidase,        beta-galactosidase, beta-galactosylceremidase,        glucosylceremidase, and acid ceremidase.    -   6. Hsp70, or a functional fragment thereof, for use as a        medicament.    -   7. Hsp70, or a functional fragment thereof, for use in the        treatment, alleviation, or prophylaxis of a lysosomal storage        disorder.    -   8. Use of item 7, wherein said lysosomal storage disorder is        selected from the group consisting of the disorders        Niemann-Pick, Gaucher, Farber, Krabbe, Fabry, and Sialidosis.    -   9. A method for increasing the uptake of a compound, said method        comprising the step of administering said compound together with        Hsp70 or a functional fragment thereof.    -   10. Method of item 9, wherein said Hsp70 or a functional        fragment thereof is covalently bound to said compound.

The invention claimed is:
 1. A method for treating Gaucher disease in ahuman comprising orally administering arimoclomol, or a pharmaceuticallyacceptable salt thereof, to the human.
 2. The method of claim 1, whereinthe human is orally administered a daily dosage of arimoclomol, or apharmaceutically acceptable salt thereof, of from about 1 ug to about100 mg per kilogram of body weight.
 3. The method of claim 2, furthercomprising administering Miglustat to the human.
 4. The method of claim2, further comprising administering enzyme replacement therapy to thehuman.
 5. The method of claim 4, wherein the enzyme replacement therapyis imiglucerase, agalsidase beta, or agalsidase alpha.